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Katherine M. Still, Cara L. Soyars, Frank Velez, Kraig S. Bower, Rose K. Sia, Denise S. Ryan, Kyle Seifert, Gordon W. Laurie, Robert L. McKown; Development of Quantitative Sandwich ELISAs for Lacritin and the Lacritin-c Splice Variant in Human Tears. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4233.
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Lacritin is a pleiotropic tear factor that promotes sustained basal tearing in rabbits (Samudre, et al., IOVS, 2011). If lacritin, and/or its receptor or effector mechanisms, are indeed downregulated in dry eye (as suggested by some mass spec studies), a sensitive sandwich ELISA could prove useful as a diagnostic assay in a recombinant lacritin rescue approach for the treatment of dry eye. Here we describe the development and testing of two lacritin sandwich ELISAs for: (i) native lacritin, and (ii) a lacritin splice variant (lacritin-c) lacking the functional C-terminal domain that might be differentially upregulated in dry eye.
Capture monoclonal anti-lacritin antibody 1F5-C9-F4 and detector polyclonal anti-N-65 antibodies were respectively generated against lacritin’s N- and C-termini. Another detector polyclonal antibody was developed against the unique C-terminus of the lacritin-c splice variant. Concentrations of detector and capture antibodies were optimized, and standard curves developed, using recombinant lacritin and lacritin-c. Each was then tested against normal human tears collected from the lower cul-de-sac using a polyester fiber rod.
1F5-C9-F4 captures both native lacritin and lacritin-c since the latter shares the same N-terminus. Anti-N-65 and anti-lacritin-c C-terminal antibodies respectively detect only native lacritin and lacritin-c. When tested with tears, lacritin appears to comprise approximately 1-2% of total tear protein, but no lacritin-c was detected; however, both are detectable by Western blotting with the same antibodies, suggesting a lower level of expression for lacritin-c in normal tears.
A sandwich ELISA has been developed for the quantitation of lacritin from small tear volumes. Although the assay does not detect tear lacritin-c, the splice variant was apparent by Western blotting. Differential upregulation of inactive lacritin-c might have deleterious effects in dry eye.
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