S0942 200tab

Mouse studies were carried out using 6–8-week-old C57BL/6 females, maintained at the animal facilities at The Scripps Research Institute. Mouse rooms are kept at 40–60% relative humidity at a temperature of 68–79 degrees F, with at least 10 room air changes per hour. Mice were injected IV with 75 µg of mAb per mouse. 16 h after injection, mice were bled and plasma was isolated. In parallel, a 384 well high binding plate (Corning 3700) was coated with anti-human IgG Fab antibody (Jackson ImmunoResearch 109-006-097) at a dilution of 1:500 and incubated overnight at 4 °C. The plate was blocked with 3% BSA in PBS for 1 h at RT. Plasma was added in a dilution series to the 384 well plate, and incubated for 1 h at RT. Detection was measured with alkaline phosphatase-conjugated goat anti-human IgG Fcγ (Jackson ImmunoResearch 109-005-008) at 1:2000 dilution in 1% BSA in PBS for 1 h. The plate was then washed and developed using a phosphatase substrate (Sigma-Aldrich, S0942-200TAB). Absorption was measured at 405 nm. Validation of antibodies relied on target specificity stated on the manufacturer’s website; independent validation was not performed. Only female mice were used in this experiment in order to maintain consistency with the liver burden assay.

Solubilized CHO cell membrane proteins (SMP) were made in house. SMP, human insulin (Sigma-Aldrich, I2643), single strand DNA (Sigma-Aldrich, D8899) were coated onto 96-well half-area high-binding ELISA plates (Corning, 3690) at 5 μg/mL in PBS overnight at 4°C. After washing, plates were blocked with PBS/3% BSA for 1 h at RT. Antibody samples were diluted at 100 μg/mL in 1% BSA with serial dilution and then added in plates and incubated for 1 h at RT. After washing, alkaline phosphatase-conjugated goat anti-human IgG Fcγ secondary antibody (Jackson ImmunoResearch, 109-055-008) was added in 1:1000 dilution and incubated for 1h at RT. After final wash, phosphatase substrate (Sigma-Aldrich, S0942-200TAB) was added into each well. Absorption was measured at 405 nm.

The antibody polyreactivity was assessed as described earlier [20 (link),49 (link)]. Solubilized CHO cell membrane protein (SMP) was coated onto 96-well half-area high-binding ELISA plates (Corning, 3690) at 5ug/mL in PBS overnight at 4°C. After washing, plates were blocked with PBS/3% BSA for 1 hour at room temperature (RT). Antibodies were diluted at 100ug/mL in 1% BSA with 5-fold serial dilution. Serially diluted samples were then added in plates and incubated for 1 hour at RT. After washing, alkaline phosphatase-conjugated goat anti-human IgG Fcy secondary antibody (Jackson ImmunoResearch, 109-055-008) was added in 1:1000 dilution and incubated for 1h at RT. After final wash, phosphatase substrate (Sigma-Aldrich, S0942-200TAB) was added into each well. The absorption was measured at 405 nm in a spectrophotometer. Den3 (Dengue-specific mAb) and Bococizumab (PCK9 antagonist) were included as benchmarking controls.