Our recommended strategy is to design campaigns with multiple immunization approaches (protein, DNA, cell-based immunogens) using a variety of WT strains and/or human-antibody producing transgenics and/or target-specific KOs to give the best chance of identifying mice that develop superior plasma titers over time. The antibody response against most targets vary depending on the method of immunization and the strain of animal used, with certain combinations of immunization approach and strain leading to maximum plasma titers. Based on our experience, we have a good sense of how plasma titers relate to hit rate, and have pre-determined ELISA and FACS target titer thresholds for advancing animals into antibody recovery.
D16 Antibody Staining
8 mice comprising 4 different strains were immunized with human and mouse Target proteins + Tolerance Breaking Protein using our HT-HOCK protocol. The first plasma titer check (D16) was performed by both ELISA and flow cytometry (M3 shown). Mice with robust titers that met both our FACS and ELISA threshold criteria were advanced to final boost, fusion, and antibody recovery. From a 4-plate screen, 18 human/mouse Target cross-reactive hits as well as 103 huTarget-specific hits were identified and confirmed. Immunizations and titer checks were continued for the remaining mice until threshold values were met, and a second group of selected mice were advanced for a second round of fusions and antibody recoveries.