Publications (3)7.44 Total impact
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Article: Slot blot immunoassay as a tool for plasmid-encoded toxin detection in enteroaggregative Escherichia coli culture supernatants.
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ABSTRACT: Plasmid-encoded toxin (Pet) is a heat-labile enterotoxin encoded in the enteroaggregative Escherichia coli (EAEC) virulence plasmid. Several evidence support the role of this 108-kDa secreted protein in the pathogenesis of EAEC diarrhea. In this study, we standardized a slot blot immunoassay for Pet detection. EAEC culture supernatants were applied onto a polyvinylidene difluoride membrane, and, using rabbit polyclonal Pet antisera, the expression of the toxin by slot blot immunoassay was observed in 9.5% of the isolates studied. In addition, no negative control reacted with Pet antiserum in this assay. This assay is a rapid, specific, reproducible, and low-cost methodology, therefore demonstrating its potential in diagnosing Pet expression. Moreover, we describe for the first time that expression of Pet can be directly detected from EAEC culture supernatants and may be used in clinical laboratorial routine instead of polymerase chain reaction detection of the pet gene, especially in developing countries where the EAEC pathotype has been considered an emerging pathogen.Diagnostic Microbiology and Infectious Disease 07/2006; 55(2):101-6. · 2.53 Impact Factor -
Article: Molecular engineering of an EGFP/disintegrin-based integrin marker.
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ABSTRACT: Disintegrins are viper venom peptides, which bind integrins with high affinity (10(-8) M) and selectivity. Among them, eristostatin (Er) selectively binds and inhibits alphaIIbbeta3 integrin function. In this work we have engineered an enhanced green fluorescence protein (EGFP)-tagged Er as an alphaIIbbeta3 biomarker to be used in bioassays involving fluorescence detectors. For this, we have first constructed an EGFP bacterial expression vector, which resulted in a 6xHis tag-coding region followed by the EGFP gene and a 3' multiple cloning site (MCS) comprising nine restriction sites. This vector, termed pAZ, was used to clone the Er gene, resulting in a 32 kDa EGFP-Er fusion protein when expressed as characterized by SDS-PAGE and Western blot. Both EGFP-Er and EGFP (expressed from the empty pAZ vector) were purified by immobilized metal affinity chromatography (IMAC) and their fluorescence was measured showing similar values, thus suggesting that the Er portion is not affecting the EGFP activity. EGFP-Er, but not EGFP selectively bound to immobilized platelets as detected by confocal microscopy indicating the preservation of Er disintegrin activity and its potential use as a marker for alphaIIbbeta3 integrin. Our data suggest the use of the pAZ vector for expressing soluble EGFP-labeled proteins and the use of EGFP-fused disintegrins as markers for integrins.Toxicon 09/2005; 46(2):178-84. · 2.51 Impact Factor -
Article: A rapid assay of the sialidase activity in species of the Bacteroides fragilis group by using peanut lectin hemagglutination.
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ABSTRACT: In this study, a novel, simple and rapid hemagglutination assay by using a peanut lectin to detect a neuraminidase activity in strains of the Bacteroides fragilis group was developed. One hundred and fourteen species of the B. fragilis group isolated from children with and without diarrhea and 15 reference strains were evaluated. Neuraminidase production was determined by using the method above described and its inhibition was observed by using galactose. The neuraminidase production was observed in 54 (84.37%) diarrhea and in 43 (86%) non-diarrhea strains. HA titers were ranged from 2 to 32. This neuraminidase assays based on PNA hemagglutination is highly sensitive, reproducible and could be used as a tool to detect the sialidase activity in anaerobic bacteria, particularly, in species of the B. fragilis group.Anaerobe 12(5-6):238-41. · 2.41 Impact Factor
