Features | Partner Sites | Information | LinkXpress
Sign In
RANDOX LABORATORIES
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



ADVANCED INSTRUMENTS
DIASYS DIAGNOSTIC SYSTEMS
KARL HECHT GMBH & CO KG
WATERS CORPORATION

Channels

Genetic Tests

view channel
Image: Histopathology of pancreatic adenocarcinoma arising in the pancreas head region (Photo courtesy of Dr. KGH/Wikimedia).

Multiple Genetic Changes Linked to Increased Pancreatic Cancer Risk

A genome-wide association study, believed to be the largest of its kind, has uncovered four regions in the human genome where changes may increase the risk of pancreatic cancer. Patients with pancreatic... Read more

Immunology

view channel

Powerful Innovations in Diagnostics Unveiled at EuroMedLab 2015

New systems presented by Roche include a new standard for immunology testing, an integrated platform for hematology diagnostics, and a high-throughput analyzer to perform HbA1c tests for blood glucose management. Also, evidence was presented for a high-sensitivity cardiac troponin test to provide earlier and more accurate... Read more

Industry News

view channel

Next Generation Sequencing Provides Responsiveness for Identifying Resistant Pathogens

According to a new market analysis, next generation sequencing (NGS) may impact the infectious diseases IVD sector by bypassing some of the downside of PCR-based testing, as NGS can provide more flexibility and responsiveness in identification of new pathogenic strains. NGS may earn its place in clinical testing as its... Read more
 
Copyright © 2000-2015 Globetech Media. All rights reserved.