Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display

Abstract

Iterative affinity selection procedures were used to isolate a number of single chain Fv (scFv) antibody fragment clones from naïve Tomlinson I + J phage display libraries that specifically recognize and bind a chemokine, monokine induced by interferon-gamma (MIG/CXCL9). MIG is an important transplant rejection/biology chemokine protein. ELISA-based affinity characterization results indicate that selectants preferentially bind to MIG in the presence of key biopanning component materials and closely related chemokine proteins. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific MIG binding capability with potential applications in transplant rejection monitoring, and other biomedical applications where detection of MIG level is important.

Antibody Fragments Directed against Different Portions of the Human Neural Cell Adhesion Molecule L1 Act as Inhibitors or Activators of L1 Function

“…The selection of scFvs binding to the extracellular domain (ecd) of human L1 was essentially as described [20]. The Tomlinson I and J libraries (Geneservice, Nottingham, United Kingdom) and the recombinantly expressed L1/ecd were used for screening.…”

“…The phagemid clones were amplified and phages were extracted as described [20]. For the production of soluble scFv proteins, 1.0 ml inocula of E. coli HB 2151 non-suppressor strain infected with a glycerol stock of an individual phage-ScFv clone was transferred into culture flasks containing 1 L 2×TY/100 µg/ml ampicillin/0.1% glucose.…”

A gold nanoparticle-single-chain fragment variable antibody as an immunoprobe for rapid detection of morphine by dipstick

“…42 The amber stop codon between cmyc tag and gIII in the scFv clone is recognized as a stop codon, and soluble scFv fusion protein is produced as a consequence. This ensures the expression of all selected clones including those in which the scFvs contain TAG stop codons (TG1 is able to suppress termination and introduce a glutamate residue at these positions).…”

Development of a Novel Human Single Chain Antibody Against EGFRVIII Antigen by Phage Display Technology

“…Since, a significant part of antigen is often desorbed from solid phase during blocking and washing processes, low antigen concentration can lead to reduction of phage yield. 15,16 Hence, in this study in order to increase the output of screening steps; we didn't reduce peptide concentration during biopanning rounds. However, for increasing the screening efficiency, incubation time of the pool phages with antigen were decreased while washing numbers were increased between screening rounds (Table 1).…”

“…Soluble scFvs enriched in the periplasmic/osmotic fractions were then purified by Ni-NTA column. 16 The purity of expressed and extracted soluble scFv was evaluated by 12% SDS-PAGE. For western blot analysis, the purified scFv (30ug/lane) was resolved by 12% SDS-PAGE and transferred onto PVDF membrane (Roche, Germany) and blocked with 5% skimmed milk overnight at 4°C.…”