Phoenix winnonlin pk pd modeling and analysis software

Three female CD-1 mice (~25 g; Charles River) were injected i.p. with Fab-N at 8 mg/kg. Blood was collected in heparinized capillary tubes at 5 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after injection. Plasma was obtained by centrifuging the samples at 2,000 × g for 5 min in a microcentrifuge and stored at −80°C until analysis. The concentrations of Fabs in the plasma samples were measured by ELISA. For this, each well of a 96-well Costar 3690 plate (Corning) was incubated with 100 ng 52SR4 scFv-Fc in 25 μL carbonate/bicarbonate buffer (pH 9.6) at 37°C for 1 h. After blocking with 150 μL 3% (w/v) BSA/PBS solution for 1 h at 37°C, the prepared plasma samples were added. Peroxidase AffiniPure F(ab’)₂ fragment goat anti-human IgG F(ab’)₂ fragment-specific pAbs (Jackson ImmunoResearch) were used for detection as described above. The concentration of the Fabs in the plasma samples was extrapolated from a four-variable fit of the standard curve. PK parameters were analyzed by using Phoenix WinNonlin PK/PD Modeling and Analysis software (Pharsight). All procedures were approved by the Institutional Animal Care and Use Committee of UF Scripps Biomedical Research and were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals.