Abstract
Developing reagents with high affinity and specificity are critical to detect the environmental hormones or toxicants. Ribosome display technology has been widely used in functional protein or peptide screening and in directed evolution of protein molecules in vitro. In this study, single-chain variable fragments (scFvs) against bisphenol A (BPA) were selected from a library constructed from splenocytes of non-immunized mice. After five rounds of selection, the selected scFvs bound to BPA with high affinity. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was introduced to screen the antibody affinity and specificity to BPA. The equilibrium dissociation constants (KDS) of one clone was 1.76μM as determined by surface plasmon resonance (SPR). This study indicated that ribosome display can isolate binders to small molecules from a non-immunized naive library without any in vivo steps and can generate recombinant antibodies efficiently and rapidly. In addition, this study provides a methodological framework for detection of small molecules using recombinant antibodies.
Keywords:
Expression; Ribosome display; Sequence; Single-chain variable fragments.
Copyright © 2013 Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Antibody Affinity
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Antibody Specificity
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Benzhydryl Compounds / analysis*
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Benzhydryl Compounds / antagonists & inhibitors
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Benzhydryl Compounds / chemistry
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Benzhydryl Compounds / metabolism
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China
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Environmental Pollutants / analysis*
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Environmental Pollutants / antagonists & inhibitors
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Environmental Pollutants / metabolism
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli / enzymology
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Escherichia coli / metabolism
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Estrogens, Non-Steroidal / analysis*
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Estrogens, Non-Steroidal / antagonists & inhibitors
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Estrogens, Non-Steroidal / metabolism
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Gene Library
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Haptens / analysis
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Haptens / chemistry
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Haptens / metabolism
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Immobilized Proteins / chemistry
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Ligands
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Mice
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Molecular Sequence Data
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Phenols / analysis*
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Phenols / antagonists & inhibitors
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Phenols / chemistry
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Phenols / metabolism
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Recombinant Proteins / analysis
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Ribosomes / metabolism*
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Sequence Alignment
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Single-Chain Antibodies / analysis*
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Single-Chain Antibodies / chemistry
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Single-Chain Antibodies / genetics
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Single-Chain Antibodies / metabolism
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Spleen / metabolism
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Surface Plasmon Resonance
Substances
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Benzhydryl Compounds
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Environmental Pollutants
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Estrogens, Non-Steroidal
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Haptens
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Immobilized Proteins
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Ligands
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Phenols
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Recombinant Proteins
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Single-Chain Antibodies
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bisphenol A