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Anna F Ablamowicz, Ashley Woodward, Jason J Nichols, Pablo Argueso; A Galectin-3-Based Slot Blot Affinity Assay for MUC16. Invest. Ophthalmol. Vis. Sci. 2017;58(8):494. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
MUC16 and galectin-3 contribute to the formation of the ocular surface glycocalyx barrier. The ability to examine the affinity between MUC16 and galectin-3 could aid in understanding the impact of ocular surface disease on the integrity of the glycocalyx. The purpose of this work was to adapt a slot blot assay to determine the relative affinity of galectin-3 for MUC16 collected from human tears.
Tear film samples of up to 15 µl were collected from each eye of 14 normal subjects and both samples were pooled for each subject. Total protein was determined for each pooled sample using the MicroBCA assay. Nitrocellulose membranes on a 48-well Bio-Dot slot microfiltration unit were loaded with 500 ng of recombinant human galectin-3 (rhGal-3), vacuum filtered, and incubated with 5, 10, and 15 µg of tear protein. One well was loaded with 5 µg of tear protein from each subject without rhGal-3 as a control. MUC16 binding was detected using the M11 monoclonal antibody by chemiluminescence and quantified by densitometry. Levels of complex N-linked oligosaccharides in tears were determined by lectin blot using Phaseolus vulgaris agglutinin (PHA-L). The Kruskal-Wallis test was performed to compare the relative amounts of MUC16 bound to rhGal-3.
The average total protein concentration obtained from each pooled sample was 2.26 ± 0.99 µg/µl (range 1.26 - 5.16 µg/µl). By slot blot, the average densitometry value for MUC16 in control samples was 6145.18 ± 2953.63 (range 1574.36 – 11901.52). The median values for the normalized relative amount of MUC16 bound to rhGal-3 for 5, 10, and 15 µg of protein were 0.67, 0.86, and 1.02. Statistical analysis to compare the relative amount of MUC16 bound to rhGal-3 with each amount of tear protein approached statistical significance (H = 5.09, p = 0.08). Lectin blot with PHA-L revealed the presence of two distinct bands with molecular weights of approximately 250 kDa in the tear fluid.
Slot blot is a viable method to determine the relative binding affinity of MUC16 to rhGal-3 using tear samples.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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