Intrasplenic immunization is used for the production of hybridomas in situations where only very small quantities of the antigen are available. Typically, it lends itself extremely well to producing antibodies to proteins that have been purified by electrophoresis and subsequent blotting onto nitrocellulose. Antibodies produced by this route are always immunoglobulin class M as only one immunization is used. This method is covered by Animal Procedures legislation in most countries as it is an invasive surgical procedure and welfare issues must be addressed.
The antigen must be in a highly aggregated or immobilized form. This technique is frequently used to produce antibodies to proteins separated by electrophoresis. The gel is stained in the normal manner and the proteins blotted over onto nitrocellulose. The band containing the protein of interest is then excised from the blot and used as the antigen. The presence of protein stains and other reagents makes little difference and as the in vivo part of the protocol is very short there are no long-term animal welfare issues.
1. Induce and maintain anesthesia using a mixture of fentanyl/ fluanisone with midazolam (Hypnorm/Hypnovel; see Note 4). The dosage for these agents is 0.25 mL of each active ingredient plus 0.5 mL of water given IP at a rate of 0.1 mL per mouse.
2. Shave the hair along the midscapular line above the position of the spleen and make a 1-cm incision made through the skin. Cut through the muscle wall to expose the spleen, and then deliver it through the opening complete with its pedicle.
3. Introduce the antigen into a pocket made in the spleen through the capsule and return the organ to the body cavity.
4. Close the muscle layer with three or four sutures and then close the skin likewise.
5. Keep the mice warm and close to a source of drinking water until they recover from the anesthetic, which takes approx 1-2 h. Generally, the mice suffer no side effects and will be feeding, grooming and showing no signs of discomfort soon after recovery from anesthesia.
6. Use the mice as donors for cell fusion 7 d after the intrasplenic immunization.
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