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Lakshman Subbaraman, Mirunalni Thangavelu, David McCanna, Lyndon Jones; Tear film cytokine analyses using a novel electrochemiluminescent array technique. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4325.
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© ARVO (1962-2015); The Authors (2016-present)
To optimize a novel, multiplex electrochemiluminescent array technique (Meso Scale Discovery, Rockville, MD) to quantify tear film inflammatory markers and to validate the signal linearity and recovery of tear samples in cytokine and chemokine assay kits.
Tear samples were collected from 10 participants over two visits and 4uL from each participant was pooled to be used for linearity and recovery analyses. 1ul of individual tears were tested using both the human pro-inflammatory 9-plex and human chemokine 9-plex assays. Manufacturer’s protocol was followed for these assays. Linearity of the signal intensity was tested using 1uL, 2uL and 3uL of pooled tear samples. Two standards (Std 3 and Std 4) from both the kits were spiked with 1uL and 2uL of pooled tears to assess recovery.
Interleukin-8 (448.8±50.8 pg/mL) and Interleukin-6 (24.4±3.1 pg/mL) were linear in the cytokine array for all volumes tested. Average concentration determined for IL6 and IL8 in tear samples were 7.9±4.3 pg/mL and 415.7±188.6 pg/mL respectively. Interleukin-10 was not detected in any of the samples and Interleukin-1B was below the lower limit of detection (LLOD). In the chemokine array, IL-8 (500.9±31.7 pg/mL), MCP -1 (400.7±79.1 pg/mL) and IP-10 (591720.1±5699.9 pg/mL) were linear at all volumes, MCP-4 and MDC were linear only at 2ul and 3ul volumes. Eotaxin-3, MIP-1B and TARC were below LLOD. Recovery was good for all chemokines except for IP 10; the endogenous signal masked the signal from the spike. Average concentration of IL8 and MCP-1 were 513.5±411.6 pg/mL and 574.3±496.8 pg/mL respectively.
A novel, multiplex electrochemiluminescent assay with superior sensitivity and recovery that provides a rapid and convenient method for quantifying tear film cytokines has been optimized. Six of the nine cytokines and three out of nine chemokines were detected in all samples tested. When using low volumes of sample in a multiplex array, signal linearity is a significant parameter to be tested to ensure that the sample tested is in the linear range of the assay. This novel technique can be employed to determine cytokine and chemokine expression in low volumes of tears collected from patients with dry eye and contact lens related dry eye.
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