The methods described are suitable for use with immunogens derived from a variety of sources, for example, bacterially expressed fusion proteins, baculovirus-expressed proteins or synthetic peptides conjugated to suitable carrier proteins (see Chapter 2). Antibody responses to peptide immunogens often differ from those where the immunogen is a larger macromolecule in that maximal antipeptide titres (which arise rapidly after two to three immunizations) do not always coincide with maximal titres against the intact protein (which tend to peak rather later at four to six immunizations). Although anti-peptide enzyme-linked immunosorbent assays (ELISAs) are useful for the monitoring of an immune response, there is no substitute for screening for immunoreactivity against the intact protein, for example, by immunoprecipitation or Western blotting.
Individual variation in antibody response is often significant, so it is advisable to immunize more than one animal (two or three rabbits per immunogen is recommended). Immune responses are generally poorer in specific pathogen-free rabbits, probably reflecting their greater immune naivity.
Adjuvants stabilize immunogens so that they induce the immune system persistently over a prolonged period. Oil-water adjuvants such as Freund's are extremely effective but must be prepared properly as stable emulsions. Such emulsions are very viscous, do not separate even after standing for long periods, and do not disperse if pipetted onto the surface of water. Well-prepared immunogens administered in Freund's adjuvant can persist for weeks and there is, therefore, little point in repeating immunizations too frequently. Freund's complete adjuvant (FCA), which contains bacterial cell wall components to induce nonspecific inflammation, potentiates the antibody response to immunogens. However, FCA can cause ulceration (resulting in loss of immunogen and discomfort to the animal) if administered in too large a bolus in one place or if given more than once. For this reason, subsequent immunizations are given using Freund's incomplete adjuvant (FIA), which contains no bacteria. An effective alternative to oil-based adjuvants is either to administer the immunogen on alum as a fine adsorbed suspension or to precipitate the immunogen with acetone. These treatments render proteins partially insoluble and thus more persistent immunogens (see Note 1).
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