Journal
ANALYTICAL BIOCHEMISTRY
Volume 488, Issue -, Pages 59-64Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2013.10.037
Keywords
Ribosome display; Single-chain variable fragments; Sequence; Expression
Funding
- National Nature Science Foundation of China [81030052]
- National Science and Technology Supporting Program [2012BAK08B06, 2012BAJ25B03-02]
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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 (scEvs) against bisphenol A (BPA) were selected from a library constructed from splenocytes of non-immunized mice. After five rounds of selection, the selected scEvs 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 1.76 mu M 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. (C) 2013 Elsevier Inc. All rights reserved.
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