Solid-phase radioimmunoassay for human neutrophil elastase: A sensitive method for determining secreted and cell-associated enzyme

As a multifunctional serine protease, human neutrophil elastase (HNE) plays critical roles in a variety of physiopathological processes, such as acute lung injury, emphysema, atherosclerosis, and arthritis. The quantification of HNE is important in many applications. In this paper, we report an aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) assay for detection of HNE using a tetramethylrhodamine (TMR)-labeled DNA aptamer probe. The affinity complex of HNE and DNA aptamer probe was well separated from the unbound aptamer probe in CE separation based on the difference of electrophoretic mobility. Broad complex peaks appeared due to possible multiple binding. The 45-mer aptamer having TMR labeling on the 40th T base was used as affinity probe, as larger complex peaks were obtained. We investigated the effects of various metal cations (Na, K, and Mg) in sample buffer on the binding of HNE and the aptamer in CE-LIF analysis. The presence of Na, K, or Mg in sample buffer caused a decrease of complex peaks, and Mg showed a larger effect. Under optimized conditions, this aptamer CE-LIF assay enabled the detection of HNE at 0.5 nM. This assay showed good specificity and allowed for detection of HNE spiked in diluted human serum sample. Graphical abstract The complex of HNE and DNA aptamer probe was isolated from the unbound aptamer probe in CE separation due to difference of electrophoretic mobility, allowing a CE-LIF assay for HNE.

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“…By far, common methods for HNE detection include immunoassays, enzyme activity assay, etc. [7][8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Detection of human neutrophil elastase by aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence using specified site fluorescently labeled aptamer

2017

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“…After a 1-hour incubation at 37"C, fluid was aspirated from the wells, and cells were removed by centrifugation. Cell-free supernatants were assayed for lactoferrin and elastase by RIA, as previously described (17,18).…”

Section: Determination Of Lipoxygenase and Cyclooxygenase Productsmentioning

confidence: 99%

Inhibition of 5‐lipoxygenase product formation and polymorphonuclear cell degranulation by tenidap sodium in patients with rheumatoid arthritis

Blackburn

Heck

Loose

et al. 1991

Arthritis & Rheumatism

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We studied the effect of tenidap sodium, a new antiinflammatory/antirheumatic drug (120 mg/day for 7 days), on eicosanoid production and neutrophil degranulation in patients with rheumatoid arthritis. Endogenous prostaglandin E, levels and ex vivo production of leukotriene B, (LTB,) were measured in synovial fluid samples obtained at baseline and 1 week later. We measured peripheral blood polymorphonuclear cell (PMN) degranulation following surface-bound IgG stimulation, a possible 5-lipoxygenase product-mediated event, by determining lactoferrin and elastase release into the culture fluid. We found decreased levels of endogenous prostaglandin E, as measured by radioimmunoassay, and decreased ex vivo production of LTB, by PMN as measured by high performance liquid chromatography, in synovial fluid samples from patients who took tenidap. Release of the granule proteins lactoferrin and elastase was decreased in PMN obtained

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Neutrophil lactoferrin content: Variation among mammals

et al. 1988

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Lactoferrin (Lf) in blood and/or marrow neutrophils was semiquantified using indirect immunofluorescence technique in nine mammalian species. Neutrophil iron-binding reactivity (NFeBR), which corresponds primarily to Lf, was also visualized and semiquantified using functional cytochemical (FeNTA-AF) technique at the light microscopic level in these nine and in an additional fifteen mammalian species, and in selected species at the ultrastructural level. Neutrophil immunoreactive Lf was positively correlated with total cellular and granule content of NFeBR among these nine species, and with previously reported concentrations of neutrophil Lf quantified by radioimmunoassay. Relative levels of Lf in neutrophil extracts from rat, hamster, and human were confirmed using SDS-polyacrylamide gel electrophoresis and immunoblotting. Relatively high levels of immunoreactive neutrophil Lf and/or NFeBR were observed in carnivores (ten species) and primates (six species). Among rodents (five species), the levels were variable, and the artiodactyls (four species) studied had low levels. These results demonstrate that neutrophil Lf levels vary widely among mammalian species. In addition, FeNTA-AF technique provides a rapid means of evaluating animals for relative quantities of neutrophil Lf.

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Solid-phase radioimmunoassay for human neutrophil elastase: A sensitive method for determining secreted and cell-associated enzyme

Cited by 14 publications

References 13 publications

Detection of human neutrophil elastase by aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence using specified site fluorescently labeled aptamer

Detection of human neutrophil elastase by aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence using specified site fluorescently labeled aptamer

Inhibition of 5‐lipoxygenase product formation and polymorphonuclear cell degranulation by tenidap sodium in patients with rheumatoid arthritis

Neutrophil lactoferrin content: Variation among mammals

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