Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody

W. D. Bedzyk, K. M. Weidner, L. K. Denzin, L. S. Johnson, K. D. Hardman, M. W. Pantoliano, E. D. Asel, E. W. Voss

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

Single-chain antibody of the (NH2) V(L)-linker-V(H) (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested C(H)1 and C(L) domain interactions may be more prevalent in V region molecular dynamics than structure.

Original languageEnglish (US)
Pages (from-to)18615-18620
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number30
StatePublished - 1990
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology

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