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Robert L McKown, Brooke M Justis, Casey E Coburn, Ronald W Raab, Bruce Rivers, Rose K Sia, Gordon W Laurie; Development of a Quantitative Multiplex Western Blot Assay for Tear Lacritin Proteins. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4914. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Lacritin is a tear glycoprotein reported to be decreased in patients with various forms of dry eye disease including Sjögren’s Syndrome dry eye disease. Human tear lacritin has been detected by Western blot analysis as an active lacritin monomer and an inactive tissue transglutaminase cross-linked polymer. A splice variant of lacritin with unique intron coding sequences and unknown functions termed lacritin-c has also been detected in human tear samples. Quantitation of the different forms of tear lacritin may provide a diagnostic tool for dry eye associated ocular diseases. Here we report a multiplex Western blot assay for the quantification of multiple lacritin forms in tear samples from healthy adults.
Tears collected from healthy individuals using a polyester fiber rod or Schirmer strips were eluted by centrifugation and total protein concentrations determined by the BCA assay. Tear proteins normalized to 200 µg/ml and purified recombinant lacritin of known concentrations were separated by SDS PAGE, transferred to nitrocellulose and challenged by anti-lacritin antibodies and unique lacritin-c antibodies. Western blots were developed by fluorescent secondary antibodies and imaged by a LI-COR Odyssey CLx imaging system. Specific protein bands for lacritin, lacritin-c, and cross-linked polymers of lacritin were quantified by pixel density. Decreasing concentrations of purified recombinant lacritin were run on each blot and quantified to generate a standard curve used to approximate concentrations of tear lacritin proteins.
Tear samples from healthy adults eluted from wicks and Schirmer strips were analyzed by multiplex Western blots in preparation for receipt of hundreds of samples from the phase 2 trial of LacripepTM in Sjogren's Syndrome Dry Eye and from a non-interventional study of lacritin from healthy volunteers. Preliminary analyses suggest that active monomeric lacritin, inactive cross-linked polymers of lacritin, and a splice variant of lacritin (lacritin-c) are present respectively at approximately 0.12, 0.09, and 0.18 µg/ml relative to recombinant lacritin standard curves (R2 > 0.98).
A multiplex Western blot assay has been developed to quantitate multiple forms of lacritin found in human tears. Validation of this assay with tears from healthy adults provides a baseline for analysis of tear lacritin proteins from diseased individuals in future studies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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