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Development of a Quantitative Immunoassay for Tear Lacritin Proteoforms
Brooke M Justis1, Casey E Coburn1, Ethan M Tyler1
1School of Integrated Sciences, James Madison University, Harrisonburg, VA USA.
Translational Vision Science & Technology
|September 4, 2020
Summary
A new immunoassay quantifies multiple lacritin proteoforms in human tears, aiding dry eye disease diagnosis. This advanced method offers superior sensitivity for tear lacritin analysis.
Area of Science:
- Ophthalmology
- Biochemistry
- Immunology
Background:
- Lacritin, a tear glycoprotein, is crucial for ocular surface homeostasis.
- Lacritin is deficient in dry eye disease tears.
- Lacritin exists as active monomer, inactive polymers, and a splice variant (lacritin-c).
Purpose of the Study:
- Develop a novel immunoassay to quantify multiple lacritin proteoforms in human tears.
- Establish a potential diagnostic tool for ocular diseases based on lacritin levels.
Main Methods:
- Tears collected on Schirmer strips were eluted.
- Protein concentration determined, followed by SDS-PAGE and Western blot.
- Detection using anti-Pep Lac N-term antibodies and fluorescent secondary antibodies.
Main Results:
- Quantified monomer, lacritin-c, and polymer proteoforms in human tears.
- Total lacritin ranged from 1.8% to 14.8% of total protein.
- Detected monomer lacritin at 6-176 µM, lacritin-c at 2-46 µM, and polymers at 1-23 µM.
Conclusions:
- Developed a highly sensitive immunoassay for multiple lacritin proteoforms.
- This assay surpasses previous methods in sensitivity and ability to distinguish proteoforms.
- Enables precise quantification of tear lacritin for clinical trial sample analysis.
