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Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing

J Biol Chem. 2015-08-01; 
Caitlin A Kowalsky, Matthew S Faber, Aritro Nath, Hailey E Dann, Vince W Kelly, Li Liu, Purva Shanker, Ellen K Wagner, Jennifer A Maynard, Christina Chan, Timothy A Whitehead
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Monoclonal Antibody Services … 3 Anti-TROP2 monoclonal antibody m7E6 (29) was produced in 293-6E cells and purified by protein A column by GenScript … mouse, and anti-rabbit HRP-conjugated secondary antibodies (Thermo Scientific) and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Dallas, TX … Get A Quote

摘要

Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness... More

关键词

Bordetella pertussis, TROP2, antibody, antibody engineering, conformational epitope mapping, epitope mapping, protein-protein interaction, tumor necrosis factor (TNF)