Immunogen Preparation and Immunization Procedures for Rats and Mice
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A high-titer antibody response usually requires use of an adjuvant for the first (priming) immunization. For most purposes, the immunogen is prepared by emulsification in a mineral oil containing heat-killed mycobacterium (Freund’s complete adjuvant/3-FCA). The emulsion ensures that the antigen is released slowly into the animal’s circulation, and the bacteria stimulate the animal’s T-helper cell arm of the immune system. Further booster (secondary) immunizations are almost always necessary for production of high antibody levels, and these are given either in phosphatebuffered saline (PBS) or as an oil emulsion (bacteria are not normally included in the boosting injections; a suitable oil adjuvant is Freund’s incomplete adjuvant/3-FIA). A large number of alternative adjuvants are available, but FCA/FIA (for priming and boosting respectively) usually produces maximal immune responses. However, FCA in particular can produce adverse effects in some cases and is not normally recommended for use in humans or primates Alum adjuvants are often chosen as an alternative and can be used in humans. Immunization with substances with molecular weights <3000 (such as peptides) are not normally immunogenic and will require conjugation to a carrier protein (see ref. 1 and Chapters 117– 119), such as purified protein derivative (PPD) or keyhole limpet hemocyanin (KLH).
KeywordsPurify Protein Derivative Keyhole Limpet Hemocyanin Glass Syringe High Antibody Level Immunization Procedure
- 1.Thorpe, R. (1994) Producing antibodies, in Immunochemistry Labfax (Kerr, M. A. and Thorpe, R., eds.), Bios Scientific, Oxford, UK, pp. 63–81.Google Scholar