Figure 1.

Protein A binding can be determined using a high-throughput assay. ScFvs were permuted based on the VH (variable region of the heavy chain) and VL (variable region of the light chain) and screened using a high-throughput assay to determine the amount of protein A binding. As expected, the heavy chain determined the amount of protein bound to protein A. F5, a scFv known to bind well to protein A, was used as a positive control. A7 refers to D1–2A7 and was used as a negative control. TS1 refers to EphA2-TS1. TS1* refers to a scFv with the EphA2-TS1 variable region and CDR-H2 of F5. This scFv had a high percentage of scFv bound to protein A resin, but lost the ability to bind to EphA2.