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K. Seifert, N. Gandia, K. S. Bower, D. S. Ryan, C. D. Coe, L. Peppers, R. L. McKown, G. W. Laurie; Development of a Protein Microarray Assay for Human Tear Lacritin. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4173.
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Dry eye is an underdiagnosed and poorly understood disease that affects the quality of life of over 25 million Americans. Lacritin is a natural human tear protein that is prosecretory, mitogenic and with cleavage, antimicrobial, and several small trials suggest that lacritin may be down-regulated in patients suffering from dry eye. Sensitive and predictive clinical assays are needed. Protein microarrays are a relatively recent development that can be conducted much like an ELISA, but uses a much smaller volume of sample. Here we describe progress towards the development of a quantitative protein microarray assay for tear lacritin.
Tears were collected from the lower cul-de-sac of healthy individuals using a polyester fiber rod (Transorb Wick, Filtrona, Richmond, VA). Rabbit antiserum made against the lacritin N-terminus was used in a protein microarray assay (BioOdyssey Calligrapher MiniArrayer, Bio-Rad) and Western blot analysis to detect and quantitate lacritin present in tears.
Preliminary protein microarray analysis suggests that the amount of lacritin present in tears from healthy individuals is approximately 7% (2.6-9.7%). This analysis is consistent with data obtained using the same antiserum in a quantitative ELISA. Western blot and densitometry analysis also suggests lacritin concentrations in this range.
Lacritin can be detected in tears using a protein microarray approach. The concentration of lacritin detected in tears is supported by both ELISA and Western blot data. Using a protein microarray approach, multiple tear samples at multiple dilutions can be assayed on a single slide with increased accuracy of the data. This assay can be used to quantitate the amount of lacritin in both healthy and disease states.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 (RLM and GWL). NIH R42 EY015376 (RLM).
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