Fluorometric cell-ELISA for quantifying rabies infection and heparin inhibition

Rincón, Verónica; Corredor, Adriana; Martínez-Gutiérrez, Marlen; Castellanos, Jaime E.

doi:10.1016/j.jviromet.2005.03.002

Cited by 9 publications

(12 citation statements)

References 19 publications

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“…Supernatant and RNA from HUVECs were obtained and stored at 280 uC until use. Activation of HUVECs was measured by quantifying ICAM-1 with a fluorometric cell-ELISA, as standardized previously (Rincó n et al, 2005). Briefly, ICAM-1 was detected with a mAb (Zymed), followed by addition of biotinylated anti-mouse IgG antibody (Vector Laboratories) and incubation with alkaline phosphatase-coupled streptavidin (Vector Laboratories) and a fluorogenic substrate (4-methylumbelliferyl phosphate; Molecular Probes).…”

Section: Methodsmentioning

confidence: 99%

Levels of soluble ST2 in serum associated with severity of dengue due to tumour necrosis factor alpha stimulation

Houghton-Triviño

Salgado

Rodríguez

et al. 2009

Journal of General Virology

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The interleukin-1 receptor-like-1 protein (IL1RL1), also known as ST2, has been shown previously to regulate T-cell function and is produced by T cells and endothelial cells. It was reported recently to be elevated in mild dengue patients during acute disease. The ST2 gene encodes several splice products: L (long), V (short) and s (soluble). A cohort of 38 patients with dengue haemorrhagic fever (DHF) and mild dengue fever (DF) were evaluated using a secreted soluble ST2 (sST2) ELISA. The RNA expression of ST2 was evaluated by real-time quantitative RT-PCR using patients' peripheral blood mononuclear cells (PBMCs) and in vitro using human umbilical vein endothelial cells (HUVECs) exposed to sera from dengue patients. DHF patients had higher levels of serum sST2, tumour necrosis factor alpha (TNF-a), interleukin (IL)-8 and IL-10 compared with DF patients and normal healthy control individuals. However, viraemia was indistinguishable between mild and severe cases. No changes in ST2 mRNA expression were found in PBMCs from these two groups of dengue patients. In vitro, sST2 was elevated in HUVECs treated with patient sera. Neutralization of TNF-a in patient sera by pre-treatment with a TNF-a antibody inhibited the upregulation of sST2 expression in HUVECs. These results implicate serum TNF-a in the modulation of expression of sST2 in an in vitro system, and indicate that sST2 could be associated with the severity of disease. Further studies to determine whether sST2 levels are predictive of the severe form of the disease and the role of sST2 in immune regulation are warranted.

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Section: Methodsmentioning

confidence: 99%

Levels of soluble ST2 in serum associated with severity of dengue due to tumour necrosis factor alpha stimulation

Houghton-Triviño

Salgado

Rodríguez

et al. 2009

Journal of General Virology

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“…After 24 h incubation, the medium was removed and replaced with medium containing serial dilutions of LOV: 1.25, 2.5, 5, 10 and 20 M . At different times (3,6,12,24,36,48 and 72 h), LOV was removed, and 50 l of MTT (0.5 mg/ml, prepared in PBS) was added to each well. After 3 h incubation at 37° in 5% CO 2 , 100 l of dimethyl sulfoxide was added to each well to solubilize the formazan crystals, and the plates were subsequently incubated for 15 min.…”

Section: Cytotoxicity Assaymentioning

confidence: 99%

Statins Reduce Dengue Virus Production via Decreased Virion Assembly

2011

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Background: Most of the effects of statins can be explained by pleiotropic effects independent of their lowering of serum cholesterol; in some cases, these effects have been shown to be a result of the role of statins in the prenylation of cellular proteins, some of which are involved in the life cycle of animal viruses. This study evaluated the potential antiviral activity of lovastatin (LOV) against dengue virus (DENV) infection of epithelial and endothelial cells (VERO cells, epithelial cells derived from African green monkey kidney, and HMEC-1 cells, human dermal microvascular endothelial cells). Methods: To evaluate its potential antiviral effects, LOV was used before, during and after inoculation of cell cultures with DENV. Results: Before and after viral inoculation, LOV caused a reduction in virus yield (80% for HMECs and 25% for VERO cells). However, with LOV treatment after inoculation induced a marked increase (2- to 9-fold) in viral-positive RNA while the amount of viral protein increased only by 13–23%. A moderate reduction (1 log unit) in viral titer occurred concurrent with the increase in DENV genomic RNA and protein within the cells. Conclusions: According to our results, LOV appears to have a greater effect on viral assembly than on replication, resulting in the cellular presence of viral genomic RNA and proteins that fail to take the normal assembly pathway.

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“…The results obtained in the studied concentration range were significant as they showed the ability of the system to measure higher analyte concentrations and thus broaden its applicability. [1][2][3][4][5] in the concentration range 0 ng mL -1 to 61.6 ng mL -1 . The average CEF and the standard deviation of the measurement is also listed.…”

Section: Methodsmentioning

confidence: 99%

“…This is because the measurement of a small absorption requires the measurement of a small difference between two large signals, corresponding to the intensity with and without sample, unlike for example fluorescence spectroscopy where the measurement can be made in principle against a zero background signal. Consequently, fluorometric detection of ELISA is typically 100 -1000 times more sensitive than colorimetric detection [3] . However, fluorometric detection requires the fluorescent labeling of some of the ELISA reagents whilst the cost of fluorometric microplate readers is significantly higher than basic 2 colorimetric microplate readers as more expensive optical components are required.…”

mentioning

confidence: 99%

Cavity-Enhanced Immunoassay Measurements in Microtiter Plates Using BBCEAS

et al. 2016

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We report on the first detailed use of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection system for immunoassay. A vertical R ≥ 0.99 optical cavity was integrated with a motorized XY stage, which functioned as a receptacle for 96-well microtiter plates. The custom-built cavity enhanced microplate reader was used to make measurements on a commercially available osteocalcin sandwich ELISA kit. A 30-fold increase in path length was obtained with a minimum detectable change in the absorption coefficient, αmin(t), of 5.3 × 10–5 cm–1 Hz–1/2. This corresponded to a 39-fold increase in the sensitivity of measurement when directly compared to measurements in a conventional microplate reader. Separate measurements of a standard STREP-HRP colorimetric reaction in microtiter plates of differing optical quality produced an increase in sensitivity of up to 115-fold compared to a conventional microplate reader. The sensitivity of the developed setup compared favorably with previous liquid-phase cavity enhanced studies and approaches the sensitivity of typical fluorometric ELISAs. It could benefit any biochemical test which uses single pass absorption as a detection method, through either the label free detection of biologically important molecules at lower concentrations or the reduction in the amount of expensive biochemicals needed for a particular test, leading to cheaper tests.

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Fluorometric cell-ELISA for quantifying rabies infection and heparin inhibition

Cited by 9 publications

References 19 publications

Levels of soluble ST2 in serum associated with severity of dengue due to tumour necrosis factor alpha stimulation

Levels of soluble ST2 in serum associated with severity of dengue due to tumour necrosis factor alpha stimulation

Statins Reduce Dengue Virus Production via Decreased Virion Assembly

Cavity-Enhanced Immunoassay Measurements in Microtiter Plates Using BBCEAS

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