Identification of Isolated Preocular Fluid Proteins

Fullard, Roderick

Abstract

Biochemical analysis of the proteins in human tear fluid has the potential to provide a great deal of in vivo information about the function of the lacrimal secretory system and about preocular film/ocular surface interactions - both in health and disease. However, research progress has been greatly limited by (a) the use of widely varying, often traumatic, tear collection techniques, (b) failure to recognize the heterogeneous origins of tear proteins and (c) identification procedures which have proven inadequate when applied to whole tears. To help overcome these problems, a new approach to the study of tear proteins is proposed. The objectives of this research are to (1) determine normal protein composition of the preocular fluid and the contribution made by each primary source, (2) isolate tear proteins prior to identification, and (3) use immunological techniques to identify the purified proteins. To achieve this, basal and reflex tears will be collected atraumatically by micropipette. Further samples will be irrigated specifically from the corneal surface using a non-contact corneal irrigation chamber. Proteins will be isolated by systematically purifying samples with high pressure liquid chromatography (HPLC) - using size exclusion, ion exchange and reversed phase techniques. Purity of protein fractions will be verified by polyacrylamide gel electrophoresis and isoelectric focusing. Isolated proteins will be screened for serum analogs by crossed immunoelectrophoresis against antisera to tears and serum. Enzyme-linked immunosorbent assays (ELISA) will be used to identify proteins in tears. Upon successful completion of this project, major RO1 funding will be sought for further studies of the purified proteins: (a) crystallization studies (by the X-Ray Crystallography CORE), and (b) production of monoclonal antibodies to tear-specific proteins. These will serve as further positive protein identifiers in studies of dry-eye patients and in immunohistochemical studies of ocular surface/tear film interactions (in conjunction with an Ophthalmologist). Successful isolation and purification of proteins from non-invasively collected tears will overcome many of the current problems and controversies which prevent the definition of normal tear protein composition.