## Cholesterol Loading and Ultrastable Protein Interactions Determine the Level of Tumor Marker Required for Optimal Isolation of Cancer Cells

We investigate how many target proteins per cell are sufficient for a cell to be isolated

Jayati Jain; Gianluca Veggiani; Mark Howarth

2013

#### Scholarcy highlights

• The ability to isolate specific cells from complex samples has been central to many areas of human biology, including immunology and stem cell biology
• We have shown how antigen number, antibody affinity, cholesterol level, and bead linkage all contribute to the ability to magnetically isolate cancer cells
• To enhance recovery of cancer cells bearing low levels of tumor antigen, these results suggest that one should load the cells in the sample for 1 hour with cholesterol, seek the highest affinity biotinylated antibody available, and capture the biotinylated antibody with streptavidin that is covalently bound to the solid phase, avoiding any intermediary weak link
• Different antibodies have previously been compared for sorting tumor cells, based on epithelial cell adhesion molecule expression, but the antibody affinity was not established
• Regular monoclonal or polyclonal antibodies are generally functional after site-specific chemical biotinylation and we showed that streptavidin-based capture of chemically biotinylated primary antibodies against HER2 or EpCAM was advantageous for cell isolation
• For the MDA-MB-453 cell line with $7-fold lower expression than BT474, the optimized conditions gave$20% improved recovery
• We found that modifying cholesterol levels and HER2 signaling had a major effect on cell isolation
• An ideal series of anti-epithelial cell adhesion molecule antibodies having a uniform binding site but a wide range of affinities was not available, but we showed that optimizing bead linkage and loading with cholesterol enhanced isolation of cells expressing low levels of EpCAM

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