Saponin-MPLA Nanoparticle Adjuvant Preparation

Saponin-MPLA nanoparticles (SMNP) adjuvant was prepared as previously described^{23 (link)}. Briefly, solutions at 20 mg/ml were prepared of cholesterol, DPPC, and PHAD MPLA (Avanti Polar Lipids) in 20% MEGA-10 detergent (Sigma). Quil-A saponin (InvivoGen) was dissolved in Milli-Q water at a final concentration of 100 mg/ml. These were mixed at a mass ratio of 10:2:1:1 (Quil-A:chol:DPPC:MPLA) and diluted in PBS to a final cholesterol concentration of 1 mg/ml. The solution was equilibrated overnight at 25 °C and then dialyzed against PBS using a 10 kDa MWCO cassette. The adjuvant was then sterile filtered, concentrated using Amicon Ultra Centrifugal Filters (50 kDa MWCO, Millipore Sigma), and purified by FPLC using a Sephacryl S-500 HR size exclusion column (Cytiva Life Sciences). The concentration was determined using a cholesterol quantification assay (Sigma Aldrich).