Production and Characterization of Antibodies Against Synthetic Peptides

Immunization protocols vary greatly among laboratories. In general, there are no hard and fast rules, and most protocols give satisfactory results. The methods described below are designed to give optimal results with minimal injury to the test animal, and we have used them extensively and successfully for several years (1 –6 ). Peptide immunizations differ from those where the immunogen is a larger macromolecule in that maximal antipeptide titers (which arise rapidly after two to three immunizations) do not always coincide with maximal titers against the intact protein (which tend to peak rather later at four to six immunizations). Thus, although antipeptide enzyme-linked immunosorbent assays (ELIS As) are useful gages of immune responsivity, there is no substitute for eventual screening on the intact protein (e.g., by immunoprecipitation, Western blotting, and so forth). Individual variation in antipeptide response is very marked, so it is advisable to use several animals (three to six) per immunogen. Rabbit responses are generally poorer in specific pathogen-free (SPF) animals, probably reflecting their greater immune naivity. Mouse responses are often best in F1 crosses (e.g., Balb/c�C57Bl/6) rather than pure strains. Alternatively, SJL mice generally respond well.