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Date of Graduation
Bachelor of Science (BS)
Department of Integrated Science and Technology
Robert L. McKown
Purpose: Lacritin is a naturally occurring human glycoprotein secreted from the lacrimal gland as a component of tears. Preliminary studies suggest that down-regulation of lacritin is associated with various ocular diseases, such as dry eye syndrome and blepharitis. Thus, lacritin shows potential as a new topical therapeutic for the treatment of ocular diseases. Previous studies have shown that recombinant human lacritin, when topically applied to rabbit eyes, promotes basal tearing. Antibodies produced against the terminal ends of human lacritin were used to develop a clinical immunodiagnostic assay that detect and quantify lacritin in human tear samples. In order to develop lacritin as a human therapeutic, it must be tested in an animal model system prior to human clinical trials. Previous studies have revealed that canines also suffer from dry eye syndrome, having clinical parallels to humans in the diagnosis and treatment of this disease. The assay used to detect lacritin in human tears was used to detect a lacritin ortholog in canine tears. The canine lacritin ortholog was cloned, sequenced, and a recombinant protein purified for the development of a canine lacritin immunoassay. This work describes the development of an immunodiagnostic ELISA assay to quantitate canine lacritin in tear samples and implementation of this assay to compare levels of canine lacritin in tears from normal and dry eye animals.
Methods: Rabbit polyclonal antibodies were produced against a synthetic peptide with the amino acid sequence of the first 20 amino acids of canine lacritin and purified over a Protein A Sepharose column. The antibodies were titrated against recombinant canine lacritin to produce an indirect Enzyme Linked Immunosorbent Assay (ELISA). The components of this assay, including the dilutions of the primary detection antibodies and secondary HRP-conjugated antibody, were optimized via checkerboard titrations. The ELISA was then used to detect and quantitate lacritin in healthy canine tear samples and tear samples from canines clinically diagnosed with dry eye. Western blot analysis was used to visualize lacritin in canine tear samples.
Results: An N-terminus peptide of amino acids identical to canine lacritin was synthesized and used to immunize rabbits (cLACRT N-Term-KLH/BSA). Titration of the 10 week anti-cLACRT NT and 10 week anti-cLACRT NT Protein A purified antisera (from rabbit 6924) detected lacritin at 0.1 to 1,000 ng, while the preimmune serum (from rabbits 6924 and 6925) did not detect lacritin. Levels of lacritin in healthy and dry eye canine tear samples were analyzed by an indirect ELISA in triplicate using the standard curve of recombinant canine lacritin to express values as nanograms of lacritin per 10 ng total tear protein. Immunoanalysis of normal and dry eye tears detected an average of 3.5 ng lacritin in normal tears and an average of 0.7 ng lacritin in tears collected from dogs diagnosed with dry eye. Western blot analysis using the 10 week anti-cLACRT NT Protein A purified antiserum detected prominent bands in healthy tears, and faint or absent bands in dry eye tears. The preimmune serum did not detect any bands.
Conclusions: An immunodiagnostic ELISA assay and Western blot analysis was successfully developed to quantitate and visualize lacritin in canine tear samples. Immunodiagnostic analysis and Western blot analysis shows that canine lacritin is down-regulated approximately 5 fold in tears from canines clinically diagnosed with dry eye compared to tears from healthy animals.
Enghauser, Alison Mae, "Development of an immunodiagnostic assay for canine tear lacritin" (2014). Senior Honors Projects, 2010-2019. 124.