Abstract
Phage display of peptides and proteins has successfully been employed to produce binding molecules of altered affinity. Little is known, however, regarding the impact on affinity measurements of phage-displayed molecules compared to their native freely soluble configuration. That identical affinities can be obtained was shown by Scatchard analysis of the native antibody, its single chain derivative (scFv), and its phage-displayed single chain counterpart for the ligand digoxin. No significant difference, within one standard deviation, was detected in affinity for digoxin when the phage-displayed scFv was compared to either its soluble scFv form or the purified antibody. In addition, no change in binding specificity was detected, within two standard deviations, when the binding proteins were challenged with two commonly cross-reactive compounds (dihydrodigoxin and digitoxin). That phage-display can be employed for molecules having high binding affinities (Kd of 6 x 10(-11) M) is also shown.
Highlights
Phage display of peptides and proteins has successfully been employed to produce binding molecules of altered affinity
Recombinant phage antibodies from well 3H showed a high rate of substrate turnover and were selected for further enrichment on digoxin-BSA covalently linked to magnetic particles
The resulting [3H)phagemid PEG concentrate had a titer of 1.2 x 1013 cfu/ml and gave positive enzyme-linked immunosorbant assay (ELISA) signal at a dilution of greater than 104. These phagemids were highly enriched for digoxin binding as evidenced by another round of individual colony rescue followed by a digoxin-specific ELISA where all the wells tested were reactive with digoxin
Summary
Fab', antigen binding fragment; scFv, single chain variable fragment of an antibody; hGH, human growth hormone; ELISA, enzyme-linked immunosorbant assay; VH, variable region of heavy chain; VL , variable region of light chain; V.. variable region ofkappa light chain; Fd, variable region and first constant region ofheavy chain; g3p, gene III minor coat protein; PCR, polymerase chain reaction; cfu, colony forming unit; PBS, phosphate buffered saline; PEG, polyethylene glycol; IPTG, isopropylthio-f3-galactoside; BSA, bovine serum albumin; bp, base pair. Human growth hormone (hGH) gene III fusion protein was shown to be folded correctly on phage by reactivity with a series of six conformationally sensitive hGH monoclonal antibodies. At equilibrium under identical conditions, we have compared the binding characteristics of a well-defined antibody directed against the hapten digoxin, its scFv counterpart, and this same scFv molecule displayed as a gene III fusion on phage. This close size approximation and rigidity of the steroid nucleus make possible the examination of the topographical features of the antibody binding site by analyzing both fine specificity (with structural analogs) and affinity for the primary ligand
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