title: The effect of sample treatment on separation profiles of tear fluid proteins: qualitative and semi-quantitative protein determination by an automated analysis system.
abstract: OBJECTIVE
  Qualitative and quantitative determination of tear fluid components is of increasing interest in ophthalmology. Until now, for diagnosis and course control of some diseases of the anterior parts of the eye, different methods for tear fluid protein analysis are available. Results can be obtained by polyacrylamide gel electrophoresis (PAGE), immunochemistry, and high-performance liquid chromatography (HPLC). A new method for protein separation, identification and semi-quantitative determination on a chip-based micro-fluidic technique is used for the first time to investigate tear fluids.
  
  
  METHODS
  Normal human reflex tears were obtained by stimulation with China mint oil and collected using glass capillary tubes. A lab-on-a-chip technology (developed by Agilent Technologies, Waldbronn, Germany, in co-operation with Caliper Technologies, Mountain View, California, USA) was used for separation and semi-quantitative determination of tear proteins. Tear fluid was separated on the Agilent 2100 Bioanalyzer in combination with the Protein 200 LabChip kit and the dedicated protein assay software. Time and temperature of the incubation with sample buffer were varied, and the influence of these parameters on protein separation profiles was studied. Tear proteins were also analysed by PAGE, and the results obtained by both methods were compared.
  
  
  RESULTS
  The different proteins of tear fluid can be separated by the Agilent 2100 Bioanalyzer method in very short time. By this method, the molecular weight as well as the concentration of proteins can be determined. Data are automatically stored in digital format and can be retrieved and shared. Results of this technology were comparable with the protein pattern obtained by PAGE. It was confirmed by both methods that, depending on incubation time of tear fluid with sample buffer and on temperature, different protein pattern can be obtained from the tears of one specimen.
  
  
  CONCLUSIONS
  Tear proteins - in contrast to serum or aqueous humour proteins - are very sensitive to changes in sample buffer temperature as well as incubation time with buffer. To obtain comparable results for tear fluid proteins, the sample buffer applied and the incubation time and temperature must be observed carefully. This can be demonstrated by both the new Agilent 2100 Bioanalyzer method and PAGE. These results are of importance when comparing tear fluid protein pattern for the diagnosis and course control of dry-eye syndrome and of other diseases of the anterior part of the eye.
authors.affiliation: University Eye Hospital, Auenbruggerplatz 4, 8036 Graz, Austria. otto.schmut@kfunigraz.ac.at
authors.forename: Otto
authors.initials: O
authors.lastname: Schmut
authors.forename: Jutta
authors.initials: J
authors.lastname: Horwath-Winter
authors.forename: Andrea
authors.initials: A
authors.lastname: Zenker
authors.forename: Gabriele
authors.initials: G
authors.lastname: Trummer
mesh_terms: D001082:Aqueous Humor / Q000737:chemistry; D001331:Automation; D002021:Buffers; D019411:Clinical Laboratory Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000302:isolation & purification*; D006801:Humans; D013666:Tears / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
chemical_list: D002021:Buffers; D005136:Eye Proteins; C042434:tear proteins