Features | Partner Sites | Information | LinkXpress
Sign In
Bio-Rad Diabetes Division
PURITAN MEDICAL
FOCUS DIAGNOSTICS, INC.

Immunofluorescence Assay Detects Virulent Escherichia coli

By Labmedica International staff writers
Posted on 15 Oct 2013
Image: Transmission electron micrograph of Escherichia coli (Photo courtesy of Elizabeth H. White).
Image: Transmission electron micrograph of Escherichia coli (Photo courtesy of Elizabeth H. White).
A sensitive and specific detection method for identifying enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) has been reported.

Among the methods for detection of virulence factor expression, immunoassays for which polyclonal and or monoclonal antibodies are raised, can be considered the first alternative to either animal use or in vitro culture cells assays.

Scientists at Instituto Butantan (Sao Paulo, SP, Brazil) developed a recombinant antibody using the single-chain variable fragment (scFv) approach. They evaluated the sensitivity and specificity of an intimin recombinant antibody (scFv-intimin) using an immunofluorescence assay. A synthetic gene was designed using the scFv-intimin. The intimin adherence-protein gene (eae) encodes for intimin, a 94-kDa outer membrane protein.

The evaluation of scFv-intimin by an indirect immunofluorescence assay test performance implied the use of eae-positive isolates displaying intimin variants, specifically typical EPEC (tEPEC), atypical EPEC (aEPEC), and EHEC, to assess test sensitivity and eae-negative and other Enterobacteriaceae isolates. The prototype tEPEC E2348/69 and enterotoxigenic (ETEC) H10407/E. coli K-12 isolates acted as positive and negative controls, respectively, for assay standardization.

Anti-histidine mouse antibody and goat anti-mouse immunoglobulin G- Fluorescein isothiocyanate (IgG-FITC, Sigma-Aldrich; St. Louis, MO, USA) conjugate were also included as immunofluorescence controls. In all isolates, the eae gene was first assayed by PCR, and the eae positive isolates were confirmed by fluorescent actin staining analysis. A drop of the manipulated bacterial suspension that had also been resuspended with scFv-intimin was placed on a glass slide and observed with a fluorescence microscope (Axioskop-Zeiss; Munich, Germany).

The scFv-intimin immunoassay detected typical EPEC, atypical EPEC, and EHEC isolates with 100% sensitivity and did not detect eae negative isolates, indicating 100% specificity. The authors conclude that immunofluorescence is an effective and rapid method, and scFv-intimin, an excellent tool for the diagnosis of diarrhea caused by EPEC and EHEC and also can be employed in case-control epidemiological surveys. The study was published on October 2, 2013, in the journal Diagnostic Microbiology and Infectious Disease.

Related Links:

Instituto Butantan
Sigma-Aldrich
Axioskop-Zeiss



DIASYS DIAGNOSTIC SYSTEMS
77 ELEKTRONIKA
KARL HECHT GMBH & CO KG

Channels

Hematology

view channel

Magnetic Resonance Improves Approach to Measuring Hemostasis Parameters

Two studies evaluating a magnetic resonance based detector for hemostasis measurements found its multiplex results to be rapid and accurate using lower blood volumes. At the International Society on Thrombosis and Hemostasis (ISTH) 2015 Congress (Toronto, Canada), T2 Biosystems, Inc. (Lexington, MA, USA) presented results... Read more

Lab Tech.

view channel
Image: Verigene System instrumentation consists of a Verigene Reader and one or multiple Verigene Processor SPs (Photo courtesy of Nanosphere).

Directed Molecular Diagnostic Tests Highlight Recent Microbiology Conference

The performance of a series of directed molecular diagnostic tests was confirmed by several studies presented at a recent microbiology conference. Nanosphere (Northbrook, IL, USA) manufactures tests... Read more

Industry News

view channel

QIAGEN Expands Diagnostics Menu in Partnership with Seegene

In a new collaboration, Seegene Inc. (Soul, Republic of Korea) will use its proprietary next-generation qPCR technology in developing new multiplex assays for the flagship platform of Qiagen NV (Venlo, Netherlands and Hilden, Germany). Seegene will develop a menu of multiplex assay panels for the modular QIAsymphony... Read more
 
Copyright © 2000-2015 Globetech Media. All rights reserved.