Antibody array characterization of inflammatory mediators in allergic and normal tears in the open and closed eye...

To evaluate the use of stationary phase protein array technology for tear analysis and to characterize the distribution of inflammatory mediators in normal and allergic tears in the open and closed eye states. Microcapillary tube collected Open (OTF) and closed eye tear fluid (CTF) samples from normals (N), from individuals with various active chronic ocular and other allergies (CA), as well as from an individual subsequent to unilateral induction of an acute allergic conjunctivitis were assayed using membrane arrays that were optimized to allow the detection of GM-CSF, ILs-1 alpha, 1 beta, 2-10, 12-13, INF gamma, MCP-1 and TNFalpha in clinical size samples. The protocol of a micro-well plate array specific for ILs-2, 4, 5, 8, 10, 12, 13, TNFalpha and INF gamma was modified to minimize the impact of tear matrix effects. This was used to carry out parallel analysis on selected samples. By optimizing the protocol as well as the composition of a membrane array it proved possible to significantly increase the signal-to-noise ratio and sensitivity of assay allowing for the detection of some inflammatory mediators into the sub-picogram range. This provided sufficient sensitivity to allow the assay of clinically obtainable size samples. Analysis revealed that OTF from most Ns contained a high level of IL-8 and faint signals if any for the other probed proteins. In contrast, OTF samples from most CA individuals with and without ocular symptoms exhibited to varying degrees detectable levels of most of the other probed entities. The difference between normal and pathological tears and the levels of signals became far more pronounced in the CTF compared to the OTF samples. Use of the micro-well plate assay kit without modification revealed two tear matrix effects that profoundly impact the ability to obtain meaningful ELISA data. Modifying the assay protocol reduces but does not eliminate these artifacts making it possible to approximate the concentration of many of the probed entities. The obtained data is consistent using both methodologies revealing elevated levels of IL-8 and other cytokines in approximately 60% of the OTF samples from the CA population. Other than a modest increase in IL-8, no change could be observed in the profile of OTF after induction of an acute allergic reaction.