Abstract
A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly4Ser)3 between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.
| Original language | English |
|---|---|
| Pages (from-to) | 715-728 |
| Number of pages | 14 |
| Journal | Applied Biochemistry and Biotechnology |
| Volume | 164 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - Jul 1 2011 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology
- Molecular Biology
Cite this
Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae. / Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi.
In: Applied Biochemistry and Biotechnology, Vol. 164, No. 6, 01.07.2011, p. 715-728.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae
AU - Sakamoto, Seiichi
AU - Pongkitwitoon, Benyakan
AU - Nakamura, Seiko
AU - Tabata, Kaori
AU - Tanizaki, Yusuke
AU - Maenaka, Katsumi
AU - Tanaka, Hiroyuki
AU - Morimoto, Satoshi
PY - 2011/7/1
Y1 - 2011/7/1
N2 - A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly4Ser)3 between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.
AB - A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly4Ser)3 between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.
UR - http://www.scopus.com/inward/record.url?scp=79960100076&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79960100076&partnerID=8YFLogxK
U2 - 10.1007/s12010-011-9168-4
DO - 10.1007/s12010-011-9168-4
M3 - Article
C2 - 21279468
AN - SCOPUS:79960100076
VL - 164
SP - 715
EP - 728
JO - Applied Biochemistry and Biotechnology
JF - Applied Biochemistry and Biotechnology
SN - 0273-2289
IS - 6
ER -