{"id":107389,"date":"2022-04-21T14:25:42","date_gmt":"2022-04-21T18:25:42","guid":{"rendered":"https:\/\/med.virginia.edu\/cell-biology\/?p=107389"},"modified":"2026-05-21T14:06:56","modified_gmt":"2026-05-21T18:06:56","slug":"development-of-a-quantitative-immunoassay-for-tear-lacritin-proteoforms","status":"publish","type":"post","link":"https:\/\/med.virginia.edu\/cell-biology\/2022\/04\/21\/development-of-a-quantitative-immunoassay-for-tear-lacritin-proteoforms\/","title":{"rendered":"Development of a Quantitative Immunoassay for Tear Lacritin Proteoforms"},"content":{"rendered":"

UVA Author:<\/strong> Gordon Laurie
\nCitation:<\/strong> Justis BM, Coburn CE, Tyler EM, et al. Development of a Quantitative Immunoassay for Tear Lacritin Proteoforms. Transl Vis Sci Technol. 2020;9(9):13. Published 2020 Aug 6. doi:10.1167\/tvst.9.9.13<\/p>\n

DOI:<\/strong> <\/a>
\nPub-Med Number<\/strong>: 32879769<\/p>\n

\n
\n
\n

Purpose:\u00a0<\/strong>Lacritin is a tear glycoprotein with pro-tearing and pro-ocular surface homeostasis activities that is selectively deficient in most dry eye tears. Proteoforms include an active monomer, inactive polymers, and a splice variant termed lacritin-c. Quantitation of the different proteoforms of tear lacritin may provide a diagnostic tool for ocular diseases. Here, we report the development of an immunoassay for the quantification of multiple lacritin proteoforms in human tear samples.<\/p>\n

Methods:\u00a0<\/strong>Basal tears collected on Schirmer test strips with anesthesia were eluted by diffusion and centrifugation under optimized conditions. Tear protein concentrations were determined, and 2.56 \u00b5g of each sample was separated by SDS-PAGE followed by western blot analysis. Blots were challenged with anti-Pep Lac N-term antibodies. Detection was with fluorescent secondary antibodies visualized by the LI-COR Odyssey CLx imaging system and quantified with standard curves of recombinant lacritin.<\/p>\n

Results:\u00a0<\/strong>The percent total lacritin (ng lacritin\/100 ng total protein) ranged from 1.8% to 14.8%. Monomer, lacritin-c, and polymer proteoform percent total protein ranged from 1.1% to 6.3%, 0.3% to 5.4%, and 0.7% to 5.7%, respectively. Monomer lacritin was detected at concentrations of 6 to 176 \u00b5M, with lacritin-c and polymer proteoforms at 2 to 46 \u00b5M and 1 to 23 \u00b5M, respectively.<\/p>\n

Conclusions:\u00a0<\/strong>This assay greatly exceeds the power and sensitivity of our prior lacritin enzyme-linked immunosorbent assay that was not capable of distinguishing monomer from polymers and lacritin-c proteoforms.<\/p>\n

Translational relevance:\u00a0<\/strong>A new method has been developed to quantitate multiple proteoforms of tear lacritin in preparation for analyses of samples from clinical trials.<\/p>\n<\/div>\n

Keywords:\u00a0<\/strong>dry eye; quantitative immunoassay; tear lacritin proteoforms.<\/p>\n<\/div>\n

Copyright 2020 The Authors.<\/p>\n

<\/div>\n","protected":false},"excerpt":{"rendered":"

UVA Author: Gordon Laurie Citation: Justis BM, Coburn CE, Tyler EM, et al. Development of a Quantitative Immunoassay for Tear Lacritin Proteoforms. Transl Vis Sci Technol. 2020;9(9):13. Published 2020 Aug 6. doi:10.1167\/tvst.9.9.13 DOI: Pub-Med Number: 32879769 Purpose:\u00a0Lacritin is a tear glycoprotein with pro-tearing and pro-ocular surface homeostasis activities that is selectively deficient in most dry […]<\/p>\n","protected":false},"author":1740,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"inline_featured_image":false,"footnotes":"","_links_to":"","_links_to_target":""},"categories":[13],"tags":[21],"class_list":["post-107389","post","type-post","status-publish","format-standard","hentry","category-featured-publications","tag-delete"],"acf":false,"yoast_head":"\nDevelopment of a Quantitative Immunoassay for Tear Lacritin Proteoforms - Department of Cell Biology<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/med.virginia.edu\/cell-biology\/2022\/04\/21\/development-of-a-quantitative-immunoassay-for-tear-lacritin-proteoforms\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Development of a Quantitative Immunoassay for Tear Lacritin Proteoforms - Department of Cell Biology\" \/>\n<meta property=\"og:description\" content=\"UVA Author: Gordon Laurie Citation: Justis BM, Coburn CE, Tyler EM, et al. 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