Antibody Array Analysis of the Distribution of Angiogenic Modulators, Growth Factors, MMPs and TH–1/TH–2 Cytokines in Normal and Pathological Tears

Abstract

Abstract: : Purpose: We recently utilized stationary phase antibody arrays (AA) to characterize the distribution of 80 low abundance proteins in pooled open and closed (OTF and CTF) eye tears (IOVS in press). In this study, we further increased the signal–to–noise ratio of the AA thereby allowing the differential analysis of low abundance proteins in individual normal and pathological tears. Methods: AA were constructed with the aims of reducing cross talk between secondary antibodies and enhancing the sensitivity of detection. Arrays were calibrated using recombinant standards achieving sensitivities in many instances in the low femtogram range. Parallel analyses were carried out using commercial micro–well plate formatted AA of many proteins employing a protocol minimizing confounding tear matrix effects. Arrays were used to characterize OTF and CTF from normals and individuals with active chronic allergic diseases (with and without overt ocular surface tissue involvement), systemic diseases and acute unilateral allergic conjunctivitis. Results: Analysis reveals that the protein profiles of tears recovered from normal individuals and those with active chronic atopic reactions were strikingly different with the pathological samples exhibiting exceptionally high levels of many TH–1/2 cytokines, MMPs, unique growth factors such as FGFb and HB–EGF that are not detected in normal tear fluid. The latter factors could drive mucin secretion. These differences were far more pronounced in CTF samples. This also proved true in tear samples recovered from individuals with chronic sinusitis without overt symptoms of ocular surface tissue involvement. In contrast, only marginal differences were observed in the distribution of cytokines in tear fluid obtained from normals and individuals with acute allergic conjunctivitis. Conclusions: AA can be constructed that are sufficiently sensitive to allow the differential screening of normal and pathological tears. These arrays demonstrate marked differences in the distribution of a wide range of bioactive proteins in tear samples from normals and individuals with active chronic allergic diseases.

Keywords: cornea: tears/tear film/dry eye • inflammation • pathology techniques