Towards High Throughput Selection of Binding Ligands: Using Flow

Phage display libraries represent a relatively easy way to generate binding ligands against a vast num-ber of different targets. Although in principle, phage display selection should be amenable to auto-mation, this has not yet been described and present selection protocols are far from high throughput. We have examined the selection process in a systematic approach and attempted to automate each individual step. Selection is carried out in the microtiter format using 24 targets as the individual selection lot size. Output is plated onto large assay trays, and a program to pick colonies in specific orders corresponding to the selection arrangement has been developed for the Qbot picking robot. This arrays clones according to the antigen they were selected against, and allows subsequent high density analysis using high density dot blots (up to 13,000 clones in the footprint of a microtiter plate). Although it proved possible to analyze such large numbers of clones using HD dot blots, it proved extremely difficult to quantify and digitize binding information. Furthermore, variations in expression levels led to non-specific binding artifacts – well-expressed clones gave binding signals which were often non-specific. Although this could be eliminated by also arraying clones on non-specific target filters, the integration of the information from the two filter types proved extremely difficult to quantify and analyze