Senior Honors Projects, 2010-current

Date of Award

Spring 2017

Document Type

Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Integrated Science and Technology

Advisor(s)

Robert McKown, Ph.D.

Abstract

Keratoconjunctivitis sicca (KCS), also known as dry-eye disease, causes a deficiency of tears in both humans and canines. Due to the lack of effective therapeutics for the treatment of dry-eye disease, there is a market potential for a novel secretion enhancing factor. Lacritin, a naturally occurring tear glycoprotein, increases basal tearing in rabbits when topically applied to the ocular surface and shows potential as a dry-eye therapeutic. This study aims to characterize Lacritin as a biomarker of dry-eye disease in canines with KCS. A total of 46 canine tear samples, 24 normal and 22 diagnosed with KCS, were obtained through a collaboration with the Virginia-Maryland College of Veterinarian Medicine and transported to James Madison University (JMU) for analysis. A bicinchoninic acid assay (BCA) was used to determine total tear protein in the samples. An indirect enzyme-linked immunosorbent assay (ELISA) was then used to determine the total tear Lacritin concentration in each sample using previously cloned and purified canine Lacritin as a standard. Total tear protein of the samples was normalized and the proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis with canine Lacritin antibodies was used to visualize Lacritin proteins and densitometry was used to determine the amount of the active Lacritin monomer (~18 kDa) band present. This study demonstrated that dry-eye samples had a significantly higher total protein concentration than normal samples, but a significantly lower relative intensity of the monomeric Lacritin band than normal samples. There was no significant difference in total Lacritin concentration between dry-eye and normal canines, as determined by the ELISA. Thus, the lower relative intensity of monomeric Lacritin present in canines with dry-eye suggests that monomeric Lacritin has potential as a biomarker of KCS, and should researched further in the future.

Included in

Biotechnology Commons

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