Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries

Abstract

Phage display of combinatorial antibody libraries is a very efficient method for selecting recombinant antibodies against a wide range of molecules. It has been applied very successfully for the generation of therapeutic antibodies for more than a decade. To increase robustness and reproducibility of the selection procedure, we developed a semi-automated selection method for the generation of recombinant antibodies from phage display libraries. In this procedure, the selection targets are specifically immobilised to magnetic particles which can then by automatically handled by a magnetic particle processor. At present up to 96 samples can be handled simultaneously. Applying the processor allows standardisation of panning parameters such as washing conditions, incubation times, or to perform parallel selections on same targets under different buffer conditions. Additionally, the whole protocol has been streamlined to carry out bead loading, phage selection, phage amplification between selection rounds and magnetic particle ELISA for confirmation of binding activity in microtiter plate formats. Until now, this method has been successfully applied to select antibody fragments against different types of target, such as peptides, recombinant or homologous proteins, or chemical compounds

Selection of Ankyrin Targeting HIV-1 Matrix and Identification of Its Binding Domain

“…The phage-displayed ankyrin library panning has been well established as a useful tool for selecting specific and high affinity binders. With the semi-automated platform, more efficient and less time-consuming phage display panning is feasible by utilizing magnetic bead nanoparticles (Konthur et al, 2010;Chin et al, 2016). This technique takes advantage of the natural high affinity between streptavidin and biotin to allow immobilization of biotinylated proteins on the streptavidin magnetic beads (Ackerman et al, 2009;Chin et al, 2016).…”

“…The panning selection was carried out with an automated magnetic-particle processor, KingFisher (Thermo Scientific, Waltham, MA) (Konthur et al, 2010;Chin et al, 2016). Briefly, phage-displayed ankyrin library was preincubated with PTM (PBS + 0.1% Tween 20 +2% skimmed milk) for one hour.…”

“…The in vivo biotinylation preparation allows for rapid and specific conjugation of MA protein with biotin carboxyl carrier protein (BCCP) tag (Tayapiwatana et al, 2006;Orrapin and Intorasoot, 2014). The semi-automated panning process allows for a more efficient and less time-consuming process compared to conventional microtiter plate panning (Konthur et al, 2010;Chin et al, 2016). The determinations of binding site on MA were analyzed using MA tri-helical fragments and pentadecapeptides scanning by indirect and competitive ELISAs, respectively.…”

Modern magnetic immunoassay: Biophysical and biochemical aspects

“…Separate stages of magnetic separation occur when magnetic particles are transferred between the wells and rodshaped magnets, coated with a plastic coating through successive seizure and release movements. Operating mode of magnetic particle processors: the rod-shaped magnet is covered by a plastic cap and moves into a solution containing suspended magnetic beads; moving slowly up and down, the beads are attracted to the cover, and by moving the covered magnet to the next position, the beads are transferred to a new solution; once the magnet is removed from the cap, the beads are slowly suspended again; the magnet head and plastic covers are raised to the starting position to proceed to the next stage of the process (Konthur et al, 2010).…”

Affinity Selection of Peptide Binders with Magnetic Beads <i>via</i> Organic Phase Separation (MOPS)

“…Compared to a 96-well microtiter plate, magnetic beads possess increased surface area, leading to a more efficient phage panning. 11,12) We combine the advantage of magnetic beads with organic phase separation to design affinity selection of peptide binders with high specificity. Other commercially available magnetic beads, such as poly-His, glutathione Stransferase and maltose-binding protein, are also applicable to MOPS; although the affinity of each tagged-protein with its respective magnetic beads in the organic solvent should be verified.…”