A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay

作者：Sakamoto Seiichi; Tanizaki Yusuke; Pongkitwitoon Benyakan; Tanaka Hiroyuki^*; Morimoto Satoshi

来源：Protein Expression and Purification, 2011, 77(1): 124-130.

DOI：10.1016/j.pep.2011.01.010

• Enzyme-linked immunosorbent assay
• Fluobody
• Fluorescence-linked immunosorbent assay
• Ginsenosides
• Green fluorescent protein
• Single-chain variable fragment

A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv.

• 出版日期2011-5