Characterization and diagnostic use of a recombinant single-chain antibody specific for the gp116 envelop glycoprotein of Yellow head virus

Sorasak Intorasoot, Hiroyuki Tanaka, Yukihiro Shoyama, Wichet Leelamanit

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Yellow head virus (YHV) is an invertebrate nidovirus that can cause mass mortality of the cultured Penaeus monodon shrimp. A single-chain variable fragment (scFv) antibody directed against the gp116 envelop glycoprotein of YHV was constructed from hybridomas. Variable heavy (VH) and light (VL) chain genes were amplified from cDNA using antibody-specific primers, linked to generate a full-length gene via a standard peptide linker, ligated into the pET28a expression vector and transformed into E. coli. The expressed insoluble scFv antibody was solubilized, purified using immobilized metal affinity chromatography and rapid refolded; final yield 1-1.5 mg/l. Solid-phase non-competitive enzyme-linked immunosorbent assay (non-competitive ELISA) determined the affinity constant (KA) to be 3.34 ± 0.38 × 108 l/mol. The sensitivity and specificity of scFv antibody was demonstrated by ELISA, dot blot and Western blot analysis. The detection limit determined by dot blot and indirect ELISA was 9 ng and 45 ng of purified YHV, respectively. Dot-blot assays revealed that the scFv antibody could detect YHV-infected shrimp at 24 h post-infection and did not cross-react with White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) proteins. The scFv antibody therefore might find application in rapid, simple and sensitive diagnostic tests to detect YHV in farmed shrimp.

Original languageEnglish
Pages (from-to)186-193
Number of pages8
JournalJournal of Virological Methods
Volume143
Issue number2
DOIs
Publication statusPublished - Aug 1 2007

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Roniviridae
Single-Chain Antibodies
Glycoproteins
Enzyme-Linked Immunosorbent Assay
Nidovirales
White spot syndrome virus 1
Penaeidae
Hybridomas
Invertebrates
Cross Infection
Affinity Chromatography
Routine Diagnostic Tests
Genes
Limit of Detection
Complementary DNA
Western Blotting
Metals
Escherichia coli
Viruses
Light

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

Characterization and diagnostic use of a recombinant single-chain antibody specific for the gp116 envelop glycoprotein of Yellow head virus. / Intorasoot, Sorasak; Tanaka, Hiroyuki; Shoyama, Yukihiro; Leelamanit, Wichet.

In: Journal of Virological Methods, Vol. 143, No. 2, 01.08.2007, p. 186-193.

Research output: Contribution to journalArticle

Intorasoot, Sorasak ; Tanaka, Hiroyuki ; Shoyama, Yukihiro ; Leelamanit, Wichet. / Characterization and diagnostic use of a recombinant single-chain antibody specific for the gp116 envelop glycoprotein of Yellow head virus. In: Journal of Virological Methods. 2007 ; Vol. 143, No. 2. pp. 186-193.
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