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

TY - JOUR

T1 - Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody

AU - Bedzyk, William D.

AU - Weidner, Karla M.

AU - Denzin, Lisa K.

AU - Johnson, Leslie S.

AU - Hardman, Karl D.

AU - Pantoliano, Michael W.

AU - Asel, Eric D.

AU - Voss, Edward W.

PY - 1990/10/25

Y1 - 1990/10/25

N2 - Single-chain antibody of the (NH2) VL-linker-VH (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 CHl and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

AB - Single-chain antibody of the (NH2) VL-linker-VH (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 CHl and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

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M3 - Article

C2 - 2211723

SN - 0021-9258

VL - 265

SP - 18615

EP - 18620

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 30

ER -