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Anna F Ablamowicz, Jason J Nichols; Tear film mucins detectable using various tear collection methods. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6178. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Both secreted mucins (MUC5AC) and membrane associated mucins (MUC16) in soluble form have been found in human tear fluid although various collection methods have been used in the literature. The purpose of this work was to determine the optimal tear collection method for analysis of tear film mucins.
Ten subjects without ocular surface disease were recruited for tear collection. Five subjects were randomly chosen to have basal tears collected using a capillary tube and the others underwent tear collection using Schirmer strips. All ten samples from the right eyes were processed separately from the left eye samples to investigate concentration of MUC5AC and MUC16, respectively. Fifteen micrograms of protein were loaded per lane into a 1% (w/v) agarose gel and run in electrophoresis buffer for two hours. After overnight capillary transfer, membranes were incubated with either MUC5AC antibody MAB2011 or MUC16 antibody OC125 for western blot analysis. Blots were developed with ECL (BioRad) and chemiluminescent signals captured with the Odyssey Fc (LI-COR). The relative amounts of MUC5AC and MUC16 were quantified with densitometry using Image StudioTM Lite and compared for statistically significant differences between tear collection methods using the Mann-Whitney test in SPSS (IBM).
Four out of ten samples analyzed for MUC5AC and nine out of ten samples analyzed for MUC16 displayed quantifiable bands. The first four lanes containing tear proteins from Schirmer strips that were analyzed for MUC5AC each showed two distinct bands, both above 460 kDa. Densitometry analysis calculating median signal intensities were 0.071 and 0.056 for the top and lower bands, respectively. Due to lack of MUC5AC bands present from the basal tears, no comparisons can be made at this time. Analysis of MUC16 median signal intensities calculated by densitometry from the Schirmer and basal groups were 0.031 and 0.054, respectively (Mann-Whitney U = 15.0, P = 0.29).
MUC5AC is present in both tears and conjunctival cells, which is apparent from two bands of differing MUC5AC electrophoretic ability captured using Schirmer strips. Although no statistically significant differences were found from this preliminary analysis, there is some evidence to support that the method chosen for tear collection may depend on the mucin of interest to optimize the amount collected.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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