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K. Seifert, A. Matherly, K. S. Bower, D. Sediq, R. L. McKown, G. W. Laurie; Development of an Immunoassay for Human Tear Lacritin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):542.
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Dry eye is a ubiquitous, underdiagnosed and poorly understood ocular surface disease that affects the quality of life of over 25 million Americans, especially women and the geriatric population. Sensitive and predictive clinical assays are needed. Lacritin is a natural human tear protein that is prosecretory, mitogenic and antimicrobial. Several small trials suggest that lacritin may be down-regulated in patients suffering from dry eye. Here we describe progress towards the development of a quantitative ELISA assay for tear lacritin.
N-terminal lacritin peptides conjugated to KLH were used as an immunogen in rabbits. Rabbit antiserum was used in a direct ELISA to detect increasing amounts of coated recombinant lacritin and lacritin present in tears from 65 healthy volunteers, of which, 14 were female, ages 23-45 (mean = 30.6), and 51 were male, ages 21-49 (mean = 33.0). Tears were collected by placing a drop of 0.5% proparacaine in the left eye. After two minutes with eyes closed, a small polyester fiber rod was placed intermittently in contact with the tear fluid in the lower cul-de-sac.
Rabbit N-terminal specific antibodies bind recombinant lacritin at high titer in a linear dilution series. Direct detection of lacritin in tears collected from healthy individuals tentatively suggests a concentration range of 0.288 - 9.870 ng/100 ng of total tear protein (mean = 4.228, std = 2.828).
Using a direct ELISA, lacritin can be detected in tear samples. This is the first step towards development of a sandwich ELISA employing N-terminal capture and C-terminal detector antibodies. Such an assay will allow documentation of the normal range of lacritin levels and lacritin levels in different forms of dry eye and other disease states.
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