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J. D. Edwards, S. Balaiya, K. V. Chalam; Estimation of TNF- Concentration in Aqueous Humor Samples of Normal Human Subjects With Bead Based Immuno Assay. Invest. Ophthalmol. Vis. Sci. 2010;51(13):711.
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Tumor necrosis factor alpha (TNF-α) is a macrophage/monocyte derived pluripotent cytokine that acts as an inflammatory mediator in a variety of ocular pathologies. In addition to its role as an inflammatory mediator, TNF-α increases with neuronal damage and remodeling. Studies have found TNF-α release from pressure damaged glial cells, suggesting a relationship with glaucomatous nerve cell damage. Evaluating TNF-α levels in glaucoma patients may also help determine disease progression and treatment control. Baseline level of TNF-α in aqueous is unknown. The purpose of the study is to determine the levels of TNF-α in normal aqueous samples.
50-100 µL of undiluted aqueous humor samples were obtained from 10 eyes (10 patients) at the time of cataract surgery. The sample collections were performed using a standard sterilization procedure. TNF-α levels were quantified using singleplex bead immunoassay (Luminex X Map, Technology) in which each spectral bead conjugated with protein/analyte specific capture antibody at specific bead region. Each analyte bound to its specific antibody followed by the detector antibody and the analyte levels were quantified by monitoring the spectral property of bead and the associated fluorescence. A known concentration of recombinant human TNF-α considered as an internal control in addition to assay standards.
A graphical plot was constructed using standard concentration against standard fluorescence. Statistical analysis was performed on this to extract the TNF-α levels in normal aqueous samples. The level of TNF-α in the normal samples were of 0.88±0.2pg/mL (Mean±Standard deviation) (p<0.001).
The TNF-α levels can be analyzed even at low levels in normal subjects using this bead immunoassay.
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