Phage Display and Selections on Purified Antigens

Abstract

The isolation of antibody fragments targeting proteins implicated in cancers and other diseases remains a crucial issue on targeted therapy or diagnostic tool development (Hoogenboom HR, Henderikx P, de Haard H. Adv Drug Deliv Rev 31 (1-2):5-31, 1998). In many case, the protein of interest, or a relevant portion of this protein such as its extracellular domain is available as purified protein. In such cases, phage display on purified antigen is an easy and fast way to select antibody fragment able to efficiently bind this antigen. However, the output of phage selection can vary significantly depending on the way to immobilize the purified antigen during selection. The following protocols describe the selection of phage antibody on purified antigen adsorbed on plastic, i.e. panning, or a selection in solution, using a biotinylated antigen as well as the corresponding screening produces, and gives hints on the advantage and drawbacks of each approach.

Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth

“…1011 phages of the Nali-H1 library were used in the first round of selection. After the third round, 186 clones were randomly picked and tested in non-absorbed Phage ELISA assay using avidin-plates and biotinylated-VAMP7 Antigen (5mg/ml) for cross-validation (Matz and Chames, 2012). Anti-VAMP7 VHH F1.1 was recovered following this screen, then mCherry-tagged anti-VAMP7 VHH F1.1 were further validated by co-expression with GFP-tagged VAMP2, VAMP4, Sec22b as control and VAMP7.…”

Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment

“…The Nali-H1 library of VHHs was screened against the recombinant biotinylated-Tau as described previously 43,48 . Non-absorbed Phage ELISA assay using avidin-plates and biotinylated-Tau Antigen (5µg/ml) was used for cross-validation of the selected clones 60 .…”

Targeting Peptides Derived from Phage Display for Clinical Imaging

“…A stepwise reduction of elution buffer pH in the final round of biopanning reduces low affinity phages, thus effectively further enriching for high affinity phages. Optimized commercial kits are also available, for example, solidphase screening, solution-sorting screening, kinetic antibody binding screening, and capture-sandwich ELISA screening [22,23].…”