TY - JOUR
T1 - A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay
AU - Sakamoto, Seiichi
AU - Tanizaki, Yusuke
AU - Pongkitwitoon, Benyakan
AU - Tanaka, Hiroyuki
AU - Morimoto, Satoshi
N1 - Funding Information:
The research in this paper was supported, in part, by the Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. This work was also funded by a Grant in Aid from the Japan Society for the Promotion of Science Asian CORE Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan .
PY - 2011/5
Y1 - 2011/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=79951942281&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79951942281&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2011.01.010
DO - 10.1016/j.pep.2011.01.010
M3 - Article
C2 - 21277981
AN - SCOPUS:79951942281
SN - 1046-5928
VL - 77
SP - 124
EP - 130
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -