  Despite the central role that antibodies play in the adaptive immune system
and in biotechnology, much remains unknown about the quantitative relationship
between an antibody's amino acid sequence and its antigen binding affinity.
Here we describe a new experimental approach, called Tite-Seq, that is capable
of measuring binding titration curves and corresponding affinities for
thousands of variant antibodies in parallel. The measurement of titration
curves eliminates the confounding effects of antibody expression and stability
that arise in standard deep mutational scanning assays. We demonstrate Tite-Seq
on the CDR1H and CDR3H regions of a well-studied scFv antibody. Our data shed
light on the structural basis for antigen binding affinity and suggests a role
for secondary CDR loops in establishing antibody stability. Tite-Seq fills a
large gap in the ability to measure critical aspects of the adaptive immune
system, and can be readily used for studying sequence-affinity landscapes in
other protein systems.
