Construction and expression of a single chain Fv fragment against pharmacologically active paeoniflorin in Escherichia coli, and its potential use in an enzyme-linked immunosorbent assay

Zhaohua Lu, Tadashi Masaki, Yukihiro Shoyama, Hiroyuki Tanaka

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

A recombinant single chain variable-fragment (scFv) antibody against paeoniflorin (PF) was produced using the hybridoma cell line C31B9. Variable regions of heavy (VH) and light (VL) chain antibody genes were directly cloned from cDNA resources of hybridoma C31B9 and assembled using splicing by overlap extension (SOE)-PCR using a (Gly4Ser)3 linker DNA. The constructed scFv genes were cloned into pET28a vectors for the generation of recombinant proteins in Escherichia coli. Most of the recombinant proteins were expressed in inclusion bodies. The yield of refolded and purified scFv was 1.89 mg per 100 mL of cell culture. The recombinant scFv displayed cross-reactivity as its mother monoclonal antibody (MAb) C31B9. Therefore, the newly expressed scFv protein was applied to quantitative ELISA to determine the total paeoniflorin (PF) and albiflorin (Alb) concentrations in peony root samples. Using PF as a standard compound, the full linear range of the assay was extended from 0.78 to 25 μg/mL. The results obtained by ELISA employing both the recombinant scFv and the original MAbC31B9 showed a reasonably good agreement with each other.

Original languageEnglish
Pages (from-to)151-155
Number of pages5
JournalPlanta medica
Volume72
Issue number2
DOIs
Publication statusPublished - Feb 1 2006

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Immunoglobulin Variable Region
Single-Chain Antibodies
Immunosorbents
Escherichia coli
Assays
Enzyme-Linked Immunosorbent Assay
Hybridomas
Recombinant Proteins
Enzymes
Genes
Paeonia
Inclusion Bodies
Cell culture
Complementary DNA
Cell Culture Techniques
Monoclonal Antibodies
Cells
Light
Cell Line
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Drug Discovery
  • Complementary and alternative medicine
  • Organic Chemistry

Cite this

Construction and expression of a single chain Fv fragment against pharmacologically active paeoniflorin in Escherichia coli, and its potential use in an enzyme-linked immunosorbent assay. / Lu, Zhaohua; Masaki, Tadashi; Shoyama, Yukihiro; Tanaka, Hiroyuki.

In: Planta medica, Vol. 72, No. 2, 01.02.2006, p. 151-155.

Research output: Contribution to journalArticle

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