High-throughput screening of single-chain antibodies using multiplexed flow cytometry

We have developed a screening method that has the potential to streamline the high-throughput analysis of affinity reagents for proteomic projects. By using multiplexed flow cytometry, we can simultaneously determine the relative expression levels, the identification of nonspecific binding, and the discrimination of fine specificities to generate a complete functional profile for each clone. The quality and quantity of data, combined with significant reductions in analysis time and antigen consumption, provide notable advantages over standard ELISA methods and yield much information in the primary screen which is usually only obtained in later screens. By combining high-throughput screening capabilities with multiplex technology, we have redefined the parameters for the initial identification of affinity reagents recovered from combinatorial libraries and removed a significant bottleneck in the generation of affinity reagents on a proteomic scale.