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Karen Dionne, Kelly Nichols, Alison McDermott, Rachel Redfern, Jason Nichols; Analysis of Tear Inflammatory Mediators: A comparison of Microarray and Luminex In Differing Subject Groups. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4324.
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© ARVO (1962-2015); The Authors (2016-present)
Inflammatory mediators may modulate dry eye disease (DED) and correlate with disease severity. This study compared two methods for detecting cytokine levels in tear samples across three subject groups.
Tears were collected from Schirmer strips of the right and left eye of 20 soft contact lens wearers (CL), 20 normal non-contact lens wearers (NOR) and 20 DED subjects and stored at -80°C. Protein was eluted and precipitated using ammonium bicarbonate and acetone. Right and left eye samples were combined for each subject. 10µg of protein was used for Quantibody Human Inflammation Array 3 (RayBiotech) and High Sensitivity Human Cytokine Kit (Millipore). Results were collected using GenePix 4000B Scanner (Molecular Devices) and Luminex MagPix reader (Millipore), respectively, and compared between instruments and subject groups.
Of the 40 analytes of the Quantibody microarray, ICAM-1, MCP-1, MIG, MCSF, TIMP-1, TIMP-2, and TNF RI had expression levels above the lower limits of the assay when all subjects were averaged. Significant differences in expression levels (p<0.05) occurred between CL and DED groups for MCSF, TIMP-1, TNF R1 between NOR and DED groups for ICAM-1, and between CL and NOR for ICAM-1, MCP-1, MCSF, TIMP-1, TIMP-2 and TNF R1. Of the 13 analytes of the Luminex assay, IL-1β, IL-4, IL-6, IL-7, and IL-8 had expression levels above the minimum detectable level when all subjects were averaged. Significant differences in expression levels (p<0.05) were only detected between CL and DED groups for IL-7 and IL-8, and between CL and NOR subjects for IL-8. The Quantibody microarray was more sensitive at detecting cytokines that were involved with lymphocytic activation and macrophage recruitment (ICAM-1, M-CSF, MCP-1), chemokine activity (MIG), tissue inhibitors (TIMP-1, TIMP-2) and cytotoxicity (TNF R1), whereas the Luminex was more sensitive at detecting immunomodulatory cytokines (IL-1b, IL-4, IL-6, IL-7) and chemokines (IL-8).
The Quantibody microarray detected more inflammatory cytokines overall in addition to detecting more significant differences between subject groups when compared to the Luminex. Differences were also noted in the types of cytokines each assay could detect from limited protein. Therefore, these factors should be considered in determining the appropriate assay for analyzing tear protein samples.
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