MAP-Chip: high-throughput single-molecule characterization of binding kinetics – MAP-Chip

Antibodies are key reagents for research, and for diagnostic, and therapeutic applications. Antibody discovery pipelines often hit a bottleneck in the validation of candidates from ultra-high throughput selections experiments (e.g., phage display).
We propose MAP-Chip, a single-molecule, high-throughput approach to simultaneously measure both association and dissociation rates of antibody candidates with high precision. Panels of antibody variants, identified by a peptide barcode, are expressed as a mixture and captured in the nanoapertures of a zero-mode waveguide array. The single-molecule binding kinetics for each candidate are then measured using fluorescently labelled antigens and associated to specific variants by reading their peptide barcode using single-molecule peptide sequencing, developed by the industrial partner. This allows demultiplexing of the measurements and association of a given phenotype (binding kinetics) to a given antibody variant. Proof of concept experiments on a panel of five nanobodies have already validated the key aspects of MAP-chip but also highlight the current limitations in terms of throughput and dynamic range. To increase throughput, we will design and test new and larger repertoires of error-resistant peptide barcodes, optimised for single-molecule peptide sequencing. To extend the dynamic range and enable the measurement of slow dissociation kinetics of high-affinity antibodies, which is currently limited by irreversible photobleaching of the bound fluorescent antigen, we will develop a dye cycling approach. Here, the antigen/antibody complex is detected dynamically at the single-molecule level by a fluorescent reporter molecule that binds the antigen with fast kinetics. We will apply MAP-Chip to characterise hits from ultra-high throughput selections, benchmarking its performance against standard bulk techniques. Finally, we will use MAP-Chip to affinity mature these hits with fine control of the kinetic parameters.