A single-chain antibody, BG4, was isolated from the Sanger phage display library (2.3 × 1010 single-chain antibody clones) through selection using a panel of intramolecular G-quadruplex structures. Two rounds of selection in solution were carried out using streptavidin-coated beads (Dynabeads M-280 Streptavidin, Invitrogen) with 1 μM of biotinylated G-quadruplex oligonucleotides for the first round of selection and 100 nM for the second round. The selected binders were then cloned into the pSANG10 expression plasmid for antibody production. Screening of the selected binders was performed by DELFIA (Dissociation-Enhanced Lanthenide Fluorescent Immunoassay) using an anti-FLAG europium-conjugated antibody (Sigma) and the DELFIA reagent (Perkin Elmer). Signal intensity was detected at 615 nm with a PHERAstar microplate reader (BMG, Labtech) using Time-Resolved Fluorescence detection.
Protocol detail
Isolation of G-Quadruplex-Binding Antibody
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Anti antibody
Antibody
Antibody production
Europium
Fluorescence
G quadruplex
Immunoassay
Library
M 280
Oligonucleotides
Phage display
Plasmid
Streptavidin
Corresponding Organization : University of Cambridge
Other organizations : Cancer Research UK
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Variable analysis
independent variables
- Number of rounds of selection (2 rounds)
- Binding of selected binders to intramolecular G-quadruplex structures (measured by DELFIA)
- Sanger phage display library (2.3 × 10^10 single-chain antibody clones)
- Biotinylated G-quadruplex oligonucleotides (1 μM for the first round and 100 nM for the second round)
- Streptavidin-coated beads (Dynabeads M-280 Streptavidin, Invitrogen)
- Anti-FLAG europium-conjugated antibody (Sigma)
- DELFIA reagent (Perkin Elmer)
- PHERAstar microplate reader (BMG, Labtech) using Time-Resolved Fluorescence detection
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