Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae

Seiichi Sakamoto, Benyakan Pongkitwitoon, Seiko Nakamura, Kaori Tabata, Yusuke Tanizaki, Katsumi Maenaka, Hiroyuki Tanaka, Satoshi Morimoto

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 languageEnglish
Pages (from-to)715-728
Number of pages14
JournalApplied Biochemistry and Biotechnology
Volume164
Issue number6
DOIs
Publication statusPublished - Jul 1 2011

Fingerprint

Single-Chain Antibodies
2,4-Dichlorophenoxyacetic Acid
Bombyx
Hemolymph
Antibodies
Larva
Acids
Assays
DNA
Enzymes
Nucleopolyhedrovirus
Immunosorbents
Herbicides
Metabolites
Peptides
Purification
2,4,5-Trichlorophenoxyacetic Acid
Enzyme-Linked Immunosorbent Assay
Cells
Hybridomas

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 journalArticle

@article{4b534e0f18fa44f79b7ae3aba7340c15,
title = "Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae",
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.",
author = "Seiichi Sakamoto and Benyakan Pongkitwitoon and Seiko Nakamura and Kaori Tabata and Yusuke Tanizaki and Katsumi Maenaka and Hiroyuki Tanaka and Satoshi Morimoto",
year = "2011",
month = "7",
day = "1",
doi = "10.1007/s12010-011-9168-4",
language = "English",
volume = "164",
pages = "715--728",
journal = "Applied Biochemistry and Biotechnology",
issn = "0273-2289",
publisher = "Humana Press",
number = "6",

}

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 -