Isolation of soluble scFv antibody fragments specific for small biomarker molecule, L-Carnitine, using phage display

J Immunol Methods. 2016 Jan;428:9-19. doi: 10.1016/j.jim.2015.11.006. Epub 2015 Dec 1.

Abstract

Isolation of single chain antibody fragment (scFv) clones from naïve Tomlinson I+J phage display libraries that specifically bind a small biomarker molecule, L-Carnitine, was performed using iterative affinity selection procedures. L-Carnitine has been described as a conditionally essential nutrient for humans. Abnormally high concentrations of L-Carnitine in urine are related to many health disorders including diabetes mellitus type 2 and lung cancer. ELISA-based affinity characterization results indicate that selectants preferentially bind to L-Carnitine in the presence of key bioselecting component materials and closely related L-Carnitine derivatives. In addition, the affinity results were confirmed using biophysical fluorescence quenching for tyrosine residues in the V segment. Small-scale production of the soluble fragment yielded 1.3mg/L using immunopure-immobilized protein A affinity column. Circular Dichroism data revealed that the antibody fragment (Ab) represents a folded protein that mainly consists of β-sheets. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific L-Carnitine binding capability with potential applications in metabolomic devices for companion diagnostics and personalized medicine applications. It may also be used in any other biomedical application where detection of the L-Carnitine level is important.

Keywords: Circular Dichroism (CD); Enzyme-linked immunosorbent assay (ELISA); Fluorescence quenching assay; L-Carnitine; Phage display; Single chain antibody fragment (scFv).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigen-Antibody Reactions
  • Biomarkers / analysis
  • Carnitine / analysis*
  • Carnitine / immunology*
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Peptide Library*
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology*
  • Single-Chain Antibodies / isolation & purification*
  • Solubility

Substances

  • Biomarkers
  • Peptide Library
  • Single-Chain Antibodies
  • Carnitine