March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Development of Quantitative Sandwich ELISAs for Lacritin and the Lacritin-c Splice Variant in Human Tears
Author Affiliations & Notes
  • Katherine M. Still
    Integrated Science and Technology,
    James Madison University, Harrisonburg, Virginia
  • Cara L. Soyars
    Biology,
    James Madison University, Harrisonburg, Virginia
  • Frank Velez
    Cell Biology, University of Virginia, Charlottesville, Virginia
  • Kraig S. Bower
    Ophthalmology, The Wilmer Eye Institute, Lutherville, Maryland
  • Rose K. Sia
    U.S. Army Warfighter Refractive Surgery Research Center at Fort Belvoir, Fort Belvoir, Virginia
  • Denise S. Ryan
    U.S. Army Warfighter Refractive Surgery Research Center at Fort Belvoir, Fort Belvoir, Virginia
  • Kyle Seifert
    Biology,
    James Madison University, Harrisonburg, Virginia
  • Gordon W. Laurie
    Cell Biology, University of Virginia, Charlottesville, Virginia
  • Robert L. McKown
    Integrated Science and Technology,
    James Madison University, Harrisonburg, Virginia
  • Footnotes
    Commercial Relationships  Katherine M. Still, None; Cara L. Soyars, None; Frank Velez, None; Kraig S. Bower, None; Rose K. Sia, None; Denise S. Ryan, None; Kyle Seifert, None; Gordon W. Laurie, EyeRx Research, Inc. (F), UVa Patent Foundation (P); Robert L. McKown, Office of Tech Transfer, JMU (P)
  • Footnotes
    Support  This research was supported by grant funding from Virginia’s Commonwealth Health Research Board (KS and RLM). NIH RO1 EY013143 and NIH RO1 EY0 18222 (GWL).
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4233. doi:
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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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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: cornea: tears/tear film/dry eye • lacrimal gland • cornea: clinical science 
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