Current methods for clinical diagnosis of dry eye syndromes are unreliable and limited by their inability to probe the underlying causes. Biochemical protein assays of the preocular fluid environment have the potential to provide specific information about causes and effects of various dry eye conditions. However, progress has been hampered by widespread use of inappropriate tear collection and biochemical assay methods. As a result, conflicting reports abound in the literature about normal tear protein levels and changes in dry eye. In the proposed research, three types of controlled, non-invasive collection methods will be used to obtain """"""""partitioned"""""""" samples from the preocular fluid environment so that protein sources can be traced. ELISA and enzyme kinetics assays, specifically designed to overcome the protein binding, inhibition and false recognition errors of less appropriate techniques, will be used for tear analysis. These assays will be applied to whole tears and batch-separated HPLC fractions to identify the sources of error in whole tear assays. In an initial study, comparing non-stimulated and stimulated tear proteins profiles in normals using established assays, the efficacy of cellulose sponges for non-stimulated tear collection will be compared with micropipettes. Further assays for key proteins will then be developed. To relate the preocular fluid protein profiles to clinical characteristics of dry eye syndromes, a combined clinical/biochemical trial will be conducted on both normals and dry eye patients. Clinical assessments will be performed by both an Optometrist and Ophthalmologist. All sample collection procedures and the full set of biochemical assays will be applied to both groups. The HPLC-purified main tear specific prealbumin (TSP) fraction will be used for (i) monoclonal antibody production and (ii) X-ray Crystallographic analysis. Other proteins suspected to be tear-specific or altered in dry eye states will be characterized biochemically and immunologically. Tear glycoproteins will be studied by lectin binding assays of protein-blotted electrophoretic fractions and by enzyme-linked lectin assay. Together these studies will help provide a more rational basis for (i) normal preocular fluid protein profiles and changes in specific dry eye states, (ii) relating biochemical changes to clinical characteristics of dry eye and (iii) understanding the functions of tear glycoproteins and tear- specific proteins and the consequences of changes in their levels.
