Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies

Abstract

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

Isolation of HCV Neutralizing Antibodies by Yeast Display

“…The method here is based on the Qiagen spin miniprep kit and is described in the literature [2]. The buffers used (P1, P2, N3, EB) are provided in the kit.…”

“…Comprehensive reviews and protocols describe yeast surface display (YSD) [1] as a powerful tool for isolating monoclonal antibodies and engineering these antibodies to fine tune their binding properties [2–5]. This protocol is focused on YSD application on the isolation of human monoclonal antibodies (HMAbs) from hepatitis C virus (HCV) infected individuals with modifications from previously described methods, which have been incorporated in three sections of the YSD system.…”

Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing

“…After adding 100 ml of additional SR-CAA, library expression was induced by the addition of galactose to a final concentration of 2%, and the yeast were grown overnight with shaking at 25°C. Two selection methods were used, one based on pulldown using ligand-coated beads (26) and the other based on FACS (13,20,21,27). For the bead-based pulldown method, ceramide-coated beads were generated by adding 10 l of 200 M ceramide-biotin (dissolved in DMSO) (Echelon Biosciences, Salt Lake City, UT) to 200 l of magnetic Dynabeads M-280 streptavidin (Invitrogen/Life Technologies, Inc.), mixing, and incubating with rotation for 10 min at 25°C.…”

“…mide-binding enriched selection output plasmid preparations as a template. The plasmids were then transformed into the yeast strain EBY100 by a lithium acetate heat shock method, and individual colonies were verified by colony PCR as described previously (27).…”

“…Individual yeast clones from the second round output were tested for binding to 200 nM ceramide by FACS as described above. Yeast clones with dramatically reduced ceramide binding were sequenced by colony PCR as described previously (27).…”