IMMUNIZATION BLEEDING SCHEDULE OF RABBITS - DOC

							IMMUNIZATION / BLEEDING SCHEDULE OF RABBITS

Injection of immunogen into rabbits in order to elicit good immune response.


Rabbits:

                 New Zealand White (NZW) Rabbit, 15-20 week-old, Female.

                 Take pre-immunization serum before antigen injection.


Immunogen Preparation
            Add Complete Freund’s Adjuvant (CFA) to the KLH-conjugated peptide.
               Homogenize for a short period on ice until a thick emulsion develops.

                 The emulsion should be very thick and not disperse when a drop of it is
                  placed on the surface of a saline solution.

                 Take up emulsion in a syringe, remove all the air bubbles.

Immunogen Injection

                 Inject subcutaneously (sc).

                 Freund’s Adjuvants are potentially harmful, be careful during preparation
                  and injection.

                 Hold the rabbit and inject the emulsion into one side near the back of the
                  neck, pinch the skin between the thumb and forefinger, pull the skin away
                  from the body and insert the needle into the space that has been created.

                 Move the end of the needle a short distance from side to side to ensure that
                  it is not inserted into muscle or the body wall.

                 Depress the plunger to inject the desired amount. Pause a few seconds. As
                  the needle is withdrawn, the puncture should be gently rubbed with the
                  forefinger and the thumb to stop any of the inoculum from escaping.

                 Move to the next site and repeat. Up to 10 sites per animal.

Boosts

                 The second injection of the same antigen mixed with equal amount of
                     incomplete Freund’s Adjuvant is performed in 3 weeks after the first
                     injection.

                 The third and any subsequent injections are done every third week.
Sampling Serum and Test Bleed

               After the third injection, the first bleed is taken 7-14 days later (see table).

                         77-day Schedule
                                   (2 bleeds)             Type          Titer         Affinity
Week 0          pre-immune bleed
                first immunization

Week 1                                                     IgM           +               +

Week 2                                                     IgM           +               +

Week 3          Second immunization                        IgG          ++              ++

Week 4                                                     IgG          ++              ++

Week 5                                                     IgG          ++              ++

Week 6          Third immunization                         IgG          +++            +++

Week 7          First bleed                                IgG          +++            +++

Week 10         Fourth immunization                        IgG          +++           ++++

Week 11         Second bleed                               IgG          +++           ++++