Cell isolation via antibody-targeted magnetic beads is a powerful tool for 
research and clinical applications, most recently for isolating circulating 
tumor cells (CTC). Nonetheless fundamental features of the cell-bead interface 
are still unknown. Here we apply a clinically relevant antibody against the 
cancer target HER2 (ErbB2) for magnetic cell isolation. We investigate how many 
target proteins per cell are sufficient for a cell to be isolated. To understand 
the importance of primary antibody affinity, we compared a series of point 
mutants with known affinities and show that even starting with subnanomolar 
affinity, improving antibody affinity improved cell isolation. To test the 
importance of the connection between the primary antibody and the magnetic bead, 
we compared bridging the antibody to the beads with Protein L, secondary 
antibody, or streptavidin: the high-stability streptavidin-biotin linkage 
improved sensitivity by an order of magnitude. Cytoskeletal polymerization did 
not have a major effect on cell isolation, but isolation was inhibited by 
cholesterol depletion and enhanced by cholesterol loading of cells. Analyzing a 
panel of human cancer cell lines spanning a wide range of expression showed that 
the standard approach could only isolate the highest expressing cells. However, 
our optimization of cholesterol level, primary antibody affinity, and 
antibody-bead linkage allowed efficient and specific isolation of cells 
expressing low levels of HER2 or epithelial cell adhesion molecule. These 
insights should guide future approaches to cell isolation, either magnetically 
or using other means, and extend the range of cellular antigens and biomarkers 
that can be targeted for CTC isolation in cancer research and diagnosis.