Protein Protocols Protein Protocols

958 Adrian
lation and may serve to protect labile antigens from degradation. Freund’s adjuvant
alone (“incomplete”) causes an inflammatory response that stimulates antibody formation,
and when made “complete” by addition of 1 mg/mL of heat-killed Mycobacterium
butyricum, this response is further enhanced. It is convenient to purchase
complete and incomplete Freund’s adjuvant ready mixed (Sigma Chemicals or
Calbiochem).
When preparing the emulsion, care should be taken to ensure that the oil remains in
the continuous phase. Injection of aliquots of the aqueous conjugate solution into the
oil via a fine bore needle, followed by repeated aspiration and ejection of the crude
emulsion, will produce the required result. An alternative for making the emulsion is to
use a homogenizer, although the generator should be retained specifically for this purpose
to avoid subsequent peptide contamination. A simple test for the success of the
preparation is to add a drop of the emulsion to the surface of water in a tube. The
emulsion should stay in a single droplet without dispersing; confirming that it is
immiscible and thus oil-phase continuous.
2.5. Choice of Animal for Immunization
Several factors need to be considered when choosing animal species for an immunization
program, including cost, ease of handling and the volumes of antisera required
(see Note 3). Small animals (such as rats and mice) have low blood volumes and present
difficulties with bleeding. Large animals such as sheep or goats are expensive to house
particularly over long periods. Rabbits or guinea pigs provide a near optimal solution,
as they are relatively cheap to house and bleeding an ear vein or cardiac puncture in
guinea pigs can provide between 10 and 30 mL of plasma from each bleed. For production
of monoclonal antibodies immunization of mice is required.
3. Methods
3.1. Carbodiimide Procedure
1. Dissolve the peptide to be coupled (400 nmol) and protein carrier (100 nmol) in a small
volume of water (