Production of human single-chain variable fragment (scFv) antibody specific for digoxin by ribosome display.
Ribosome display was applied in vitro to select single-chain variable fragment (scFv) antibody specific for digoxin from a human non-immune naive scFv library. A cell-free system was used to produce stable antibody-ribosome-mRNA (ARM) complexes to provide the linkage of genotype and phenotype, allowing simultaneous selection of a desired antibody together with its encoding mRNA. The mRNA was then recovered and amplified as DNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Repeating the display cycle enriched the selected molecules, enabling rare species to be isolated. In this study, digoxin-binding segments were selected over four cycles of ARM display and the selected DNA was cloned and expressed as a single-chain variable fragment antibody (the best scFv, A3) in Escherichia coli. The affinity (equilibrium dissociation constant Kd) of digoxin was 8.3 x 10(-8) M for A3, which validated construction of the naïve library and the power of ribosome display lending to the evolution of functional characteristics, such as potency of leading candidate antibodies to provide therapeutic antibodies. A3 was purified using affinity chromatography and determined by Western blot. The results indicate that ribosome display technnique can be efficiently used to isolate specific antibody fragments from a naive library.
Yan
Xiang-Hua
XH
Key Laboratory of Animal Molecular Nutrition, Ministry of Education, Feed Science Institute, College of Animal Science, Zhejiang University, Hangzhou, PR China. xhyan@zju.edu.cn
Xu
Zi-Rong
ZR
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004077:Digoxin / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes
D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012333:RNA, Messenger; D004077:Digoxin