Electrophoresis combined with immunologic identification of human tear proteins

The protein content of normal human tears from five subjects was examined by molecular weight separation using SDS-polyacrylamide gel electrophoresis (PAGE) and by charge separation using agarose isoelectric focusing (IEF) gels. After separation, specific proteins were identified by immunoblot and immunofixation. Tear proteins examined included albumin, IgA, IgG, prealbumin, lactoferrin, lysozyme, secretory component and transferrin. These techniques required 1 to 14 microliters unconcentrated tears. We found SDS-PAGE superior to agarose IEF to examine total tear protein pattern, and silver stain almost ten-fold more sensitive than Coomassie blue stain. Immunologic staining markedly enhanced protein detection in all tear samples and appeared to offer the definitive method to probe for a specific protein in tears. In this study prealbumin and a portion of the IgG were present in normal tears at higher than expected molecular weight, suggesting they were present in complexed form. Prealbumin and secretory component staining showed marked variability between subjects. These techniques should be applicable to examine tear proteins in a variety of ocular disease states.