Bacterial expression and characterization of an anti-desipramine single-chain antibody fragment

Gaofeng Lin, Paul R Pentel, Weilin L. Shelver, Daniel E. Keyler, Catherine A. Ross, Yoko Hieda, Michael C. Flickinger, Christopher A Pennell, Michael P Murtaugh

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Tricyclic antidepressant toxicity is a leading cause of death from intentional drug overdose. Monoclonal antibody Fab' fragments specific for the tricyclic antidepressant, desipramine, reverse acute drug toxicity but may themselves have adverse effects at therapeutic doses. To evaluate the characteristics of smaller antibody fragments, we cloned, expressed and characterized a 26 kD single chain Fv fragment (G5-sFv). A DNA sequence encoding V(H)-linker-V(L) was constructed from hybridoma mRNA encoding a high affinity monoclonal desipramine-specific IgG1 and expressed in E. coli. G5-sFv was produced at high levels as insoluble inclusion bodies. Single chain Fv was solubilized, folded in a redox buffer and affinity purified on desipramine-Sepharose. The affinity of G5-Fv for desipramine was similar to that of the corresponding monoclonal Fab' as measured by surface plasmon resonance (Fab' 5.5 ± 0.5 x 108M-1, sFv 2.3 ± 0.5 x 108 M-1). G5-sFv administered to rats after a tracer dose of 3H-desipramine produced rapid and marked redistribution of drug from tissues into serum G5-sFv was stable at 4°C for greater than 6 months but lost activity at higher temperatures. We conclude that desipramine-specific-single chain Fv expressed in E. coli retains the affinity of the parent antibody for desipramine. The pharmacokinetic effect of G5-sFv on desipramine Society for Immunopharmacology.

Original languageEnglish (US)
Pages (from-to)729-738
Number of pages10
JournalInternational Journal of Immunopharmacology
Volume18
Issue number12
DOIs
StatePublished - Dec 1996

Keywords

  • Antibody
  • Immunotherapy
  • Immunotoxicology
  • Overdose
  • Tricyclic antidepressant
  • sFv fragment

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