Extraction and Characterization of Vaccine Antigens from Water-in-Oil Adjuvant Formulations
Water-in-oil adjuvants are currently undergoing experimental testing in human vaccine trials (1‱10). Two such adjuvants are the squalene-based Montanide® ISA 720 and the mineral oil-based Montanide ISA 51 (11). Vaccines containing these adjuvants are intended to provide a lasting depot effect, with vaccine persisting at the injection site for many months (12). Because it is often convenient to formulate these vaccines well ahead of use, and because of the extended residence at the injection site, their stability must be established. Therefore, studies should be designed that will address stability at 4°C (intended storage temperature) and 37°C (body temperature) to ensure that the antigens remain intact and the vaccines consequently remain potent. With the addition of benzyl alcohol, the emulsion is broken, and the antigens are released and recoverable in the resulting aqueous layer. Storage at 4°C and 37°C of fresh formulations of ISA 720 and ISA 51 containing either of two recombinant malarial proteins, followed by extraction at various time points and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, and Western blotting will be used to illustrate the methods involved in monitoring the stability of protein antigens in these water-in-oil formulations.
KeywordsVortex Formaldehyde Agarose Electrophoresis Histidine
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