TITLE

Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin

AUTHOR(S)
Sakamoto, Seiichi; Taura, Futoshi; Pongkitwitoon, Benyakan; Putalun, Waraporn; Tsuchihashi, Ryota; Kinjo, Junei; Tanaka, Hiroyuki; Morimoto, Satoshi
PUB. DATE
April 2010
SOURCE
Analytical & Bioanalytical Chemistry;Apr2010, Vol. 396 Issue 8, p2955
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly4Ser)2 between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL−1) was improved to eightfold higher than that in conventional ELISA (0.2 μg mL−1), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.
ACCESSION #
48746708

 

Related Articles

  • Effect of some physico-chemical conditions on an immunoassay for viable Escherichia coli. Jurkevica, Agnese; Halsall, H. Brian; Seliskar, Carl J.; Heineman, William R. // Analytical & Bioanalytical Chemistry;Aug2010, Vol. 397 Issue 7, p3133 

    Viable Escherichia coli can be detected by an immunoassay in which live bacteria captured on antibody-coated paramagnetic beads are induced to synthesize the enzyme β-galactosidase, which catalyzes the hydrolysis of the slightly fluorescent substrate 4-methyl umbelliferyl-β- d-galactoside...

  • Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat. Yong-Ping Ma; Ya-Ning Hao; Wei Tang; Rong-Rong Wang; Fa-Ping Yi; You-Quan Bu; Lu-Yu Zhang; Fang-Zhou Song // International Journal of Clinical Medicine;Sep2012, Vol. 3 Issue 5, p387 

    The LTB of enterotoxigenic Escherichia coli (ETEC) expressed in Bifidobacterium infantis (BI) has been testified as mucosal adjuvant with co-vaccination BI-CfaB (the major fimbrial subunit) together in vivo in our previous study. In order to investigate the mucosal adjuvant effect of BI-LTB to...

  • Identification of Binding Epitope for Anti-Rabies Virus Glycoprotein Single-Chain Fv Fragment FV57. Tie-jun Gu; Wei Wei; Ye Duan; Chun-lai Jiang; Yan Chen; Xiang-hui Yu; Jia-xin Wu; Yong-ge Wu; Wei Kong // Protein & Peptide Letters;Nov2011, Vol. 18 Issue 11, p1099 

    Single-chain Fv fragment (scFv) of anti-rabies glycoprotein (G protein) has been recommended as a new agent for detecting and neutralizing lethal rabies virus. In this study, we constructed scFv that corresponded to the FV fragment of CR57, a monoclonal antibody against rabies virus, and called...

  • Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. Matsumoto-Takasaki, Ayano; Horie, Jinichiro; Sakai, Keiko; Furui, Yoshihiro; Sato, Reiko; Kawakami, Hiroko; Toma, Kazunori; Takayanagi, Atsushi; Shimizu, Nobuyoshi; Fujita-Yamaguchi, Yoko // BioScience Trends;Jun2009, Vol. 3 Issue 3, p87 

    T-antigen (Galβ1-3GalNAc-Thr/Ser) also known as Thomsen-Friedenreich (TF) antigen is the core 1 structure of O-linked mucin type glycans. In normal epithelium, the disaccharide structure is masked by terminal carbohydrate moieties, but is uncovered in most primary and metastatic epithelial...

  • Comparison of a Commercial Reversed Passive Latex Agglutination Assay to an Enzyme Immunoassay for the Detection of Shiga Toxin-Producing Escherichia coli. Carroll, K. C.; Adamson, K.; Korgenski, K.; Croft, A.; Hankemeier, R.; Daly, J.; Park, C. H. // European Journal of Clinical Microbiology & Infectious Diseases;Nov2003, Vol. 22 Issue 11, p689 

    A multicenter study was performed to compare the performance of a prototypic reversed passive latex agglutination assay (VTEC Screen “Seiken”; Denka-Seiken, Japan) with the Premier EHEC Enzyme Immunoassay (Meridian Diagnostics, USA) for the detection of Shiga toxin in 554 diarrheal...

  • Diagnostic Efficacy of Lsa63 Antigen for Human Leptospirosis. Alizadeh, Safar Ali; Eshraghi, Seyyed Saeed; Pourmand, Mohammad Reza; Naserpour, Taghi; Abdollahpour, Gholamreza; Rahimiforoshani, Abbas; Najafipour, Reza // Iranian Red Crescent Medical Journal;Mar2014, Vol. 16 Issue 3, p1 

    Background: Timely diagnosis of leptospirosis is essential for early and effective treatment, for there are many differential diagnoses for it.. Leptospiral researchers have an increasing interest in developing new serological methods with recombinant antigens to improve the Leptospirosis...

  • Antigen-Binding Properties of Monoclonal Antibodies Reactive with EBNA1 and Use in Immunoaffinity Chromatography. Duellman, Sarah J.; Burgess, Richard R. // PLoS ONE;2009, Vol. 4 Issue 2, p1 

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG1) and two mAbs are isotype IgM....

  • EVALUATION OF A MONOCLONAL ANTIBODIES PANEL DIRECTED AGAINST FIMBRIAL ANTIGENS K88, K99, F41 AND 987P OF ENTEROTOXIGENIC ESCHERICHIA COLI. Junco, J.; Campal, A.; Miranda, A.; Castro, M.; Arteaga, N.; Casas, S.; León, L.; Alvarez, T.; Sanchez, D.; Fernández, G.; Fontirroche, G. // Revista VacciMonitor (Vacunología y Temas Afines);Oct2002, Vol. 11 Issue 4, p1 

    Enterotoxigenic Escherichia coli (ETEC) represents a large group of strains bacteria that are toxigenic to small intestine of neonates in a number of species including pigs, cattle, sheep, and humans. It well established that the ability of ETEC to colonize the intestine is due to fimbrial...

  • Serum reactivity against bacterial pyruvate dehydrogenase: Increasing the specificity of anti-mitochondrial antibodies for the diagnosis of primary biliary cirrhosis. Miyakawa, Hiroshi; Tanaka, Atsushi; Selmi, Carlo; Hosoya, Naomi; Mataki, Norikazu; Kikuchi, Kentaro; Kato, Takashi; Arai, Junya; Goto, Toshihiro; Gershwin, M. Eric // Clinical & Developmental Immunology;Jun-Dec2006, Vol. 13 Issue 2-4, p289 

    Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated...

Share

Read the Article

Courtesy of VIRGINIA BEACH PUBLIC LIBRARY AND SYSTEM

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics