Identification of high-affinity phage-displayed VH fragments by use of a quartz crystal microbalance with dissipation monitoring

Abstract

[EN] Phage display has become a powerful tool for antibody discovery in a wide variety of fields. This technology allows specific binders for a given antigen to be selected from combinatorial libraries. A key step in the process is characterizing and evaluating antibody clones thus selected to reliably identify the best antigen binders. Novel characterization methods can provide essential insight into the binding mechanism and supplement the information obtained with conventional techniques. In this work, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to determine the kinetic and thermodynamic binding parameters for phagedisplayed VH antibody fragments. Phytohemagglutinin (PHA), a legume lectin of analytical interest, was used as a complex model antigen to select specific VH fragments from a phage-displayed library. Eight VH fragments with a unique amino acid sequence were identified as PHA binders by using the well-established enzyme-linked immunosorbent assay (ELISA). QCM-D measurements, structural analysis and principal component analysis (PCA) were used to evaluate the antibody fragments and identify clone clusters with similar binding characteristics and molecular interaction mechanisms. This unprecedented study has enabled the identification of highaffinity phage-displayed VH antibody fragments for PHA, which could be useful for PHA analysis (apparent association constant ranged from 108 to 1010 M-1). In fact, the proposed methodology provides a useful tool for evaluating and characterizing antibody fragments with capabilities beyond those of conventional techniques