Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes.
As antibody-based diagnosis and therapy grow at an increased pace, there is a need for methods which rapidly and accurately determine antibody-antigen interactions. Here, we report a method for the multiplex determination of antibody epitopes using bacterial cell-surface display. A protein-fragment library with 10(7) cell clones, covering 60 clinically-relevant protein targets, was created and characterized with massively parallel sequencing. Using this multi-target fragment library we determined simultaneously epitopes of commercial monoclonal and polyclonal antibodies targeting PSMA, EGFR, and VEGF. Off-target binding was observed for one of the antibodies, which demonstrates the methods ability to reveal cross-reactivity. We exemplify the detection of structural epitopes by mapping the therapeutic antibody Avastin. Based on our findings we suggest this method to be suitable for mapping linear and structural epitopes of monoclonal and polyclonal antibodies in a multiplex fashion and could find applicability in serum profiling as well as other protein-protein interaction studies.
School of Biotechnology, Alba Nova University Center, Royal Institute of Technology, Stockholm, Sweden.
Elton P
EP
Hudson
Mathias
M
Uhlen
Johan
J
Rockberg
D000906:Antibodies / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D000068258:Bevacizumab; D003429:Cross Reactions; D003546:Cysteine Endopeptidases / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D066246:ErbB Receptors / Q000276:immunology; D015723:Gene Library; D057166:High-Throughput Screening Assays; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013210:Staphylococcus / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology
D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D000939:Epitopes; D010446:Peptide Fragments; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab; C512478:EGFR protein, human; D066246:ErbB Receptors; D003546:Cysteine Endopeptidases