Virginie Jakob1, Livia Brunner1, Christophe Barnier-Quer1, Molly Blust2, Nicolas Collin1, Lauren Carter2, Darrick Carter3, Kelly M Rausch4, Christopher B Fox5. 1. Vaccine Formulation Laboratory, University of Lausanne, Epalinges, Switzerland. 2. Infectious Disease Research Institute, Seattle, WA, USA. 3. Infectious Disease Research Institute, Seattle, WA, USA, and Department of Global Health, University of Washington, Seattle, WA, USA. 4. Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA. 5. Infectious Disease Research Institute, Department of Global Health, University of Washington, 1616 Eastlake Ave E Ste 400, Seattle, WA 98102, USA.
Abstract
OBJECTIVES: Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. METHODS: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. RESULTS: The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. CONCLUSIONS: These methods may be useful for improving characterization approaches of antigen-adjuvant mixtures by SDS-PAGE.
OBJECTIVES: Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. METHODS:Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. RESULTS: The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. CONCLUSIONS: These methods may be useful for improving characterization approaches of antigen-adjuvant mixtures by SDS-PAGE.
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