Anti pcsk9 neutralizing antibody

All chemical reagents were obtained from Sigma-Aldrich unless otherwise specified. Anti-PCSK9 neutralizing antibody was from BPS Biosciences (San Diego, CA). Alirocumab was from Sanofi (Paris, France) and Regeneron Pharmaceuticals, Inc. (Tarrytown, NY). Evolocumab was from Amgen (Thousand Oaks, CA). Trastuzumab was from Genentech (South San Francisco, CA). All luminescence readings were performed on a Glomax Discover Detection System (Promega Corp., Madison, WI). EC₅₀ calculations were performed using GraphPad Prism (version 6.03).

LgBiT-LDLR-expressing HEK293 cells were plated at 10,000 cells/well in a 96-well plate in 100 µl of DMEM complete media (containing 10% FBS) and incubated in a cell culture incubator (37°C, 5% CO₂, humidified atmosphere) overnight. Then the medium was removed and replaced with Nano-Glo Luciferase Assay Substrate diluted into Opti-MEM. To a subset of wells, Opti-MEM was added that contained PCSK9-SmBiT, for a final concentration of 0.8 µg/ml; other wells received Opti-MEM only for NanoGlo substrate background analysis. Luminescence was monitored every minute for 30 min. Then anti-PCSK9 neutralizing antibody (BPS Biosciences) was spiked into a subset of wells, for a final concentration of 15 nM, and luminescence readings were resumed.

LgBiT-LDLR-expressing HEK293 cells were plated at 10,000 cells/well in a 96-well plate in 100 µl of DMEM complete media (containing 10% FBS), and incubated in a cell culture incubator overnight. Then the medium was removed and replaced with 25 µl of Opti-MEM (Thermo Fisher Scientific), containing 2× PCSK9-SmBiT for a final concentration of 0.8 µg/ml and 25 µl of Opti-MEM containing a 2× antibody titration. Alirocumab and evolocumab were tested starting at 60 nM, and anti-PCSK9 neutralizing antibody (BPS Biosciences) and nonspecific control antibody, trastuzumab, starting at 20 nM. The reaction was incubated for 1 h; then 12.5 µl of 5× Nano-Glo Luciferase Assay Substrate (Promega) diluted into Opti-MEM was added to the reaction. The luminescence was measured after a 15 min incubation.