Selection of Recombinant Antibodies from Antibody Gene Libraries

Abstract

Antibodies are indispensable detection reagents for research and diagnostics and represent the biggest class of biological therapeutics on the market. In vitro antibody selection systems offer many advantages over animal-based technologies because the whole selection process is independent of the in vivo immune response. In the last two decades antibody phage display has evolved to the most robust and widely used method and has already yielded thousands of antibodies. The selection of binders by phage display is also referred to as "panning" and based on the specific molecular interaction of antibody phage with an immobilized antigen thus allowing the enrichment and isolation of antigen-specific monoclonal binders from very large antibody gene libraries. Here, we give detailed protocols for the selection of recombinant antibody fragments from antibody gene libraries in microtiter plates.

A human scFv antibody generation pipeline for proteome research

“…The bacteria were centrifuged for 20 min at 10,000 × g. Phage particles in the supernatant were precipitated with 1/5 volume of 20% (w/v) polyethylene glycol (PEG)/2.5 M NaCl solution for one hour on ice with gentle shaking and pelleted by centrifugation for one hour at 10,000 × g at 4 • C. The precipitated phage were resuspended in 10 mL phage dilution buffer (10 mM TrisHCl pH7.5, 20 mM NaCl, 2 mM EDTA), 1/5 volume of PEG solution was added and incubated on ice for 20 min, followed by centrifugation for 30 min at 10,000 × g and 4 • C. The pellet was resupended in 1 mL phage dilution buffer. Residual bacteria and cell debris were removed by additional centrifugation for 5 min at 16,000 × g at 20 • C. The supernatants containing the antibody phage were stored at 4 • C. Phage titration was done as described before (Hust et al, 2007a). The scFv display rate of the packaged libraries was controlled by 10% SDS-PAGE, Western-blot and antipIII immunostain (mouse anti-pIII 1:2000, goat anti-mouse IgG AP conjugate 1:10,000).…”

“…The supernatant containing the eluted scFv phage was transferred into a new tube. 10 L of eluted scFv phage were used for titration as described before (Hust et al, 2007a). The remaining scFv phage were reamplified as described before (Hust et al, 2007a) and used for the next panning round.…”

“…10 L of eluted scFv phage were used for titration as described before (Hust et al, 2007a). The remaining scFv phage were reamplified as described before (Hust et al, 2007a) and used for the next panning round. The second and third panning round using the amplified phage were performed with the following modifications: the amount of antigen was reduced and washing cycles during panning were increased to 20, respectively 30.…”

Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells

“…The generation of potent antibodies for research, diagnosis and therapy has been facilitated by the progress in methods for building and selecting large antibody libraries [1, 2]. In addition to conventional heavy/light chain antibody libraries, there has been a growing interest in single domain antibody (sdAb) libraries.…”

Single-step Colony Assay with Autoinduction of scFv Expression for the Screening of Antibody Libraries

“…Libraries with a vast repertoire and efficient screening are two prerequisites for obtaining monoclonal antibodies with high affinity to the antigen [2,3]. Two approaches for screening currently exist: display strategy and repertoire cloning strategy [4,5].…”