Abstract
Abstract: :
Purpose: To adapt sandwich ELISA microarray (MA) technology for tear analysis and to characterize the relative distribution of a wide range of bioactive proteins in tear samples recovered during the diurnal cycle. Methods: Reflex, open and closed eye tear samples (RTF, OTF and CTF, respectively) were obtained by capillary tube and centrifuged. Membrane–bound and microwell plate formatted MA kits were evaluated for tear analysis. Based on initial findings the protocols were modified, on a trial and error basis to reduce confounding tear matrix effects and to increase the signal to noise ratio. This allowed the screening of matched RTF and pooled OTF and CTF samples for more than 80 trace proteins. Results: Modifying the blocking and detecting protocol of a commercially available membrane–bound MA system greatly increased the sensitivity of the ELISA assays allowing the tentative identification and determination of the relative distribution of >20 growth factors, cytokines, chemokines and angiogenic modulators in tear samples. The concentrations of most of these entities increased markedly in CTF. This finding is consistent with either an ocular surface tissue or inflammatory cell origin. Other proteins appeared to be of lacrimal gland origin. A prime example is angiogenin that was found in surprisingly high concentrations in all tear samples. This finding is compatible with the anti–microbial properties of this protein and a role in the mucosal defense system (Nat Immunol. 2003:269–73). Use of a microwell plate MA proved challenging due to confounding tear matrix effects. These arise from the presence in tears of a sticky factor that exhibits a high affinity for the well matrix resulting in high background reading and the well–known presence of an antibody blocking factor(s). By modifying the protocol, matrix effects were minimized and semi–quantitative data was obtained on the distribution of TIMP–1, HGF, FGFB, Ang2, Tpo, VEGF, KGF and PDGF in tears. These results complimented the membrane MA data.Conclusions: MA data reveals that tears contain a wide diversity of bioactive trace proteins that undergo a profound shift in distribution with prolonged eye closure. Results suggest that with further refinement MAs may provide a powerful tool allowing the screening of tears for a wide range of trace proteins and the quantification of a smaller number of targeted proteins.
Keywords: lacrimal gland • cornea: tears/tear film/dry eye • protein purification and characterization