A fluorescent single domain antibody against plumbagin expressed in silkworm larvae for fluorescence-linked immunosorbent assay (FLISA)

Seiichi Sakamoto, Benyakan Pongkitwitoon, Kaori Sasaki-Tabata, Waraporn Putalun, Katsumi Maenaka, Hiroyuki Tanaka, Satoshi Morimoto

研究成果: Contribution to journalArticle査読

9 被引用数 (Scopus)

抄録

A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL-1) compared with conventional ELISA (0.2 g mL -1). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.

本文言語英語
ページ(範囲)2056-2063
ページ数8
ジャーナルAnalyst
136
10
DOI
出版ステータス出版済み - 5 21 2011

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy
  • Electrochemistry

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