Novel Immuno-FRET Assay Method for Bacillus Spores and Escherichia coli O157:H7

Bruno, John G.; Ulvick, Sydney J; Uzzell, Gary L; Tabb, Joel; Valdes, Erica R.; Batt, Carl A.

doi:10.1006/bbrc.2001.5669

Cited by 16 publications

(7 citation statements)

References 14 publications

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“…Awide variety of approaches have been used including polarization (Klein et al, 1993;Fiore et al, 1988;Dandliker and de Saussure, 1970), resonance energy transfer (RET) (Morrison, 1988;Ullman et al, 1976) and gated "time-resolved" assays based on long lived lanthanide emission (Diamandis, 1988;Lövgren and Pettersson, 1990;Soini, 1984). There is continued development of new approaches to fluorescence immunoassays, including time-resolved assays (Ozinskas et al, 1993;Nithipatikom and McGown, 1987), RET assays for larger molecular weight species (Bruno et al, 2001) and multi-photon excitation (Baker et al, 2000). In recent years, there has been an emphasis on high-throughput immunoassays using multi-well plates (Schobel et al, 2001) and microparticles (Yang et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Immunoassays based on directional surface plasmon-coupled emission

Matveeva¹,

Gryczyński²,

Gryczyński³

et al. 2004

Journal of Immunological Methods

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We described a new approach to immunoassays using surface plasmon-coupled emission (SPCE). Fluorescence is visually isotropic in space, so that the sensitivity is limited in part by the light collection efficiency. By the use of SPCE, we can efficiently collect the emission and convert it to a cone-like directional beam in a glass substrate. SPCE is the coupling of excited fluorophores with a thin metal film, resulting in radiation of surface plasmons into the higher refractive index media. We used SPCE to develop a model affinity assay using labeled goat anti-rabbit immunoglobulin G (IgG) antibodies against rabbit IgG bound to a 50-nm-thick silver film. Binding of labeled IgG to the surface resulted in increased intensity observed at an angle of 75 degrees from the normal in the glass substrate. The SPCE intensity depends on proximity of the fluorophore to the silver film and does not require a change in quantum yield upon binding. The use of SPCE is shown to provide background suppression because excited fluorophores distant from the silver film do not result in SPCE. Sensitivity and selectivity can be further increased by excitation under conditions of surface plasmon resonance (SPR) because the evanescent field is enhanced by the resonance interaction and excitation is limited to the region near the metal. We believe SPCE will provide a new technology for high sensitivity and selectivity in surface-bound assays and microfluidic systems.

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Section: Introductionmentioning

confidence: 99%

Immunoassays based on directional surface plasmon-coupled emission

Matveeva¹,

Gryczyński²,

Gryczyński³

et al. 2004

Journal of Immunological Methods

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“…This is potentially quite important for the rapid, homogenous, and portable, albeit presumptive, detection of a key bioterrorist or biowarfare agent (namely anthrax) or related foodborne Bacillus pathogens [15,27]. Other fluorescence resonance energy transfer (FRET) systems for Bacillus spore detection have been devised with antibodies [27], but these systems involved Fig. 4 a and b represent fluorescence spectra for the BAS-6F and 6R aptamer beacons at 1 μg/ml in PBS (pH 7.2) in the presence of serial ten-fold dilutions of stock B. cereus spores (stock~3×10 7 spores per ml).…”

Section: Discussionmentioning

confidence: 99%

Development of Aptamer Beacons for Rapid Presumptive Detection of Bacillus Spores

Bruno

Carrillo

2012

J Fluoresc

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A library of 92 DNA aptamer sequences was developed against Bacillus anthracis (nonpathogenic Sterne strain) spores and anthrose sugar immobilized on magnetic beads. The selected DNA sequences were studied for similarities and potential binding pockets between the B. anthracis spore and anthrose aptamers. Several recurring loop structures were identified and tested for their potential to act as aptamer beacons when labeled with TYE 665 dye on their 5' ends and Iowa Black quencher on their 3' ends. Of these candidate sequences, two beacons designated BAS-6F and BAS-6R emerged which gave strong fluorescence responses at high spore concentrations (greater than 30,000 spores/ml). These aptamer beacons also detect B. cereus and B. thuringiensis spores with greater fluorescence intensity, but do not strongly detect vegetative cells from an array of other bacterial species. BAS-6F and 6R are also not capable of detecting pure anthrose, thereby probably ruling that epitope out as a spore surface target for these particular beacons. While not extremely sensitive, the BAS-6F and 6R aptamer beacons are potentially valuable for rapid presumptive detection of anthrax or Bacillus spores in suspect powders or bioterrorist activity where spore concentrations are anticipated to be high. The sequence similarities of these beacons to other published Bacillus spore aptamers are also discussed.

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“…Kenworthy et al 27 used antibodies linked to GPI-anchored proteins to characterize plasma membrane microdomains. Bruno et al 28 developed a highly sensitive assay for Bacillus spores and E. coli O157:H7 by measuring decreases in FRET activity. Additionally, Sharma et al 29 used antibodies against Torsin A and ␣-Synuclein to demonstrate the association between these proteins in a form of inherited Parkinson's disease.…”

Section: Discussionmentioning

confidence: 99%

Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy

et al. 2003

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Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. One of the principal determinants of morbidity and mortality following TBI is traumatic axonal injury (TAI). Current hypotheses on the pathogenesis of TAI involve activation of apoptotic cascades secondary to TBI. While a number of studies have demonstrated direct evidence for the activation of apoptotic cascades in TAI, the precise pathway by which these cascades are initiated remains a subject of intense investigation. As axolemmal disruption with the subsequent intra-axonal influx of large molecular weight species has been demonstrated to occur in relation to local axonal breakdown, attention has focused on cascades that may occur as a result of loss of ionic homeostasis. One proposed pathway by which this has been hypothesized to occur is the Ca(2+)-mediated activation of calmodulin and subsequent activation of the phosphatase calcineurin with dephosphorylation of a protein known as BAD, leading to a proapoptotic interaction between BAD and the mitochondrial protein Bcl-xL. While this pathway is an intriguing route for traumatic axonal pathogenesis, neither conventional immunocytochemical/histochemical nor ultrastructural approaches have had the capacity to shed insight on whether BAD and Bcl-xL interact in TAI in vivo. We describe the implementation of confocal and two-photon excitation fluorescence resonance energy transfer (FRET) microscopy techniques through which we demonstrate interaction between the proapoptotic protein BAD and the prosurvival protein Bcl-xL within TAI following TBI. Further, we report on a method to reliably detect protein interactions within aldehyde fixed tissue sections through conventional immunohistochemical approaches.

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Novel Immuno-FRET Assay Method for Bacillus Spores and Escherichia coli O157:H7

Cited by 16 publications

References 14 publications

Immunoassays based on directional surface plasmon-coupled emission

Immunoassays based on directional surface plasmon-coupled emission

Development of Aptamer Beacons for Rapid Presumptive Detection of Bacillus Spores

Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy

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