Mega 10 detergent

The saponin adjuvant used in this study is an ISCOMs-like self-assembled nanoparticle comprised of cholesterol, phospholipid, and Quillaja saponin as previously described (22 (link)). Briefly, under sterile conditions, the following solutions were made: 0.5 mL of 20 mg/mL of cholesterol (700000P, Avanti) in 20% MEGA-10 detergent (D6277, Sigma Aldrich), 0.5 mL of DPPC (850355C, Avanti) in 20% MEGA-10 detergent, and 0.5 mL of 100 mg/mL Quil-A adjuvant (vac-quil, Invivogen) in deionized H₂0 were prepared. DPPC solution was mixed with the cholesterol followed by the addition of Quil-A saponin in rapid succession. This mixture was diluted with PBS to a concentration of 1 mg/mL cholesterol and 2% MEGA-10, prior to overnight equilibration at 25°C. The lipids/saponin/surfactant solution was then dialyzed against PBS using a 10 kDa MWCO membrane for 5 days at 25°C and filter sterilized using a 0.2 Supor syringe filter. For further purification, the adjuvant solution was concentrated using 50 kDa MWCO Amicon Ultra-filters (UFC905008, Millipore Sigma) and purified by size exclusion chromatography using a Sephacryl S-500 HR size exclusion column. For quality control, the final saponin adjuvant was characterized by Limus Amebocyte Lystae assay (QCL-1000, Lonza) for low endotoxin levels. The adjuvant concentration was determined using a cholesterol quantification kit (MAK043, Sigma).