title: Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.
abstract: Single chain variable fragments (scFvs) against diethylstilbestrol (DES) were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM) complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3) for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of one clone (30-1). The measured K(D) was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.
authors.affiliation: Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Sun
authors.forename: Baoan
authors.initials: B
authors.lastname: Ning
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.forename: Xianjun
authors.initials: X
authors.lastname: Gao
authors.forename: Xianjun
authors.initials: X
authors.lastname: Fan
authors.forename: Jianqing
authors.initials: J
authors.lastname: Liu
authors.forename: Zhixian
authors.initials: Z
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D004054:Diethylstilbestrol / Q000276:immunology / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004785:Environmental Pollutants / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D004968:Estrogens, Non-Steroidal / Q000276:immunology / Q000302:isolation & purification*; D005260:Female; D005822:Genetic Vectors; D007118:Immunoassay / Q000379:methods*; D046911:Macromolecular Substances / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D012270:Ribosomes / Q000276:immunology; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000276:immunology; D020349:Surface Plasmon Resonance
chemical_list: D004785:Environmental Pollutants; D004968:Estrogens, Non-Steroidal; D046911:Macromolecular Substances; D019151:Peptide Library; D057127:Single-Chain Antibodies; D004054:Diethylstilbestrol