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PI Murray, K Wloka, G Cheung, S Rauz, OM Durrani, M Salmon, SJ Curnow; Multiplex Bead Analysis of Cytokines and Chemokines in Aqueous Humour . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1534.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To establish a multiplex bead based technique for analysis of cytokines and chemokines in control and inflammatory aqueous humour (AqH). Methods: AqH samples were collected from patients with untreated active uveitis and controls undergoing routine cataract surgery. The samples were centrifuged and the cell-free supernatant frozen at -80ºC. Samples of 50µl were incubated with a cocktail of LuminexTM beads, each coated with an individual monoclonal antibody, followed by biotinylated polyclonal antibodies and streptavidin-phycoerythrin. The fluorescence intensity of each bead population was measured using a Luminex100TM. The concentration of each molecule was determined from a standard curve of known concentrations of recombinant molecules. Results: From a single 50µl sample it was possible to determine the concentration of at least 15 different molecules. The molecules that have been measured to date are IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 (p70), TNFα, GM-CSF, G-CSF, RANTES, MCP-1, Eotaxin, IFNγ, and VEGF. Preliminary results indicate that inflammatory AqH shows distinct patterns of these molecules, while the majority are deficient in control AqH. Conclusion: The analysis of many cytokines and chemokines in AqH samples from an individual patient has previously been impossible due to the small volumes available. We have shown that multiplex bead analysis can circumvent this problem allowing a highly detailed profile of each AqH sample that can be related to the clinical and cytological data available. This technology will greatly enhance our understanding of inflammatory eye disease by allowing a detailed analysis of the relationship between these molecules and disease pathology.
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