Coyle P K, Sibony P A, Johnson C
Department of Neurology, SUNY, Stony Brook, New York 11794.
Invest Ophthalmol Vis Sci. 1989 Aug;30(8):1872-8.
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.
通过使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（PAGE）进行分子量分离以及使用琼脂糖等电聚焦（IEF）凝胶进行电荷分离，对五名受试者的正常人泪液蛋白质含量进行了检测。分离后，通过免疫印迹和免疫固定鉴定特定蛋白质。检测的泪液蛋白质包括白蛋白、免疫球蛋白A（IgA）、免疫球蛋白G（IgG）、前白蛋白、乳铁蛋白、溶菌酶、分泌成分和转铁蛋白。这些技术需要1至14微升未浓缩的泪液。我们发现，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳在检测泪液总蛋白质模式方面优于琼脂糖等电聚焦，并且银染比考马斯亮蓝染色灵敏近十倍。免疫染色显著增强了所有泪液样本中的蛋白质检测，似乎为探测泪液中的特定蛋白质提供了决定性方法。在本研究中，前白蛋白和一部分免疫球蛋白G以高于预期分子量的形式存在于正常泪液中，表明它们以复合形式存在。前白蛋白和分泌成分染色在受试者之间显示出显著差异。这些技术应适用于检测各种眼部疾病状态下的泪液蛋白质。
