Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, 54 Xianlie Road, Guangzhou, 510060, People's Republic of China.
Graefes Arch Clin Exp Ophthalmol. 2010 Jun;248(6):853-62. doi: 10.1007/s00417-009-1275-3. Epub 2010 Feb 2.
Quantitative determination of tear proteins is critical to our understanding of those ocular diseases with tear protein changes, but remains technically complex due to the small sample volumes available from patients. The aim of this study was to efficiently quantify the tear proteins by SDS-PAGE with an internal standard protein in small tear volumes.
Schirmer test paper and capillary tubes were used to collect tear samples. Soybean trypsin inhibitor (SBTI) was used as an external standard or an internal standard to analyze tear samples in 15% SDS-PAGE gel. The total tear protein and its major components were quantified by band densitometry. Total tear protein concentrations were also measured by Bradford assay. Using this internal standard method, we compared differences between tear samples collected by the Schirmer test paper and capillary tube, and then examined the differences detected between tear samples obtained from young and elderly people.
Using SBTI as an internal standard in SDS-PAGE, the total tear protein concentrations were determined to be 12.03 +/- 0.45 mg/ml, showing no difference from the result determined by the Bradford assay (P > 0.05). The quantities of the eight major tear protein bands were 2.26 +/- 0.07(18.8%), 0.10 +/- 0.01(0.8%), 0.63 +/- 0.13(5.3%), 0.52 +/- 0.07(4.3%), 1.14 +/- 0.18(9.5%), 1.33 +/- 0.21(11.1%), 2.76 +/- 0.16(23.0%), and 2.95 +/- 0.13 mg/ml (24.5%), similar to the result obtained by using SBTI as an external standard (P > 0.05). Comparing the methods of collection, we identified that the Schirmer test paper sampling induced increased concentrations of band 2 (mainly HSA) in tears, and decreased five other major tear protein bands. Comparing total protein concentrations among different age groups, we noted a higher total protein concentration in young people (P < 0.05). This high protein level was contributed equally by all major tear protein components.
Using SBTI as an internal standard, we can simultaneously quantify total tear protein and the major tear protein components by SDS-PAGE densitometry using small volume tears. This method appears promising for use as a diagnostic tool for identifying the occurrence of ocular diseases with tear protein changes.
定量测定泪液蛋白对于我们了解那些泪液蛋白发生变化的眼部疾病至关重要，但由于从患者获得的样本量较小，技术上仍然很复杂。本研究的目的是通过 SDS-PAGE 用内标蛋白从小的泪液样本中有效地定量测定泪液蛋白。
使用 Schirmer 试纸和毛细管收集泪液样本。大豆胰蛋白酶抑制剂（SBTI）被用作外部标准或内标来分析 15％SDS-PAGE 凝胶中的泪液样本。通过带密度测定法定量测定总泪蛋白及其主要成分。还通过 Bradford 测定法测量总泪蛋白浓度。使用这种内标方法，我们比较了 Schirmer 试纸和毛细管收集的泪液样本之间的差异，然后检查了年轻人和老年人泪液样本之间检测到的差异。
使用 SBTI 作为 SDS-PAGE 的内标，确定总泪蛋白浓度为 12.03±0.45mg/ml，与 Bradford 测定法的结果无差异（P＞0.05）。八个主要泪蛋白条带的数量为 2.26±0.07（18.8％），0.10±0.01（0.8％），0.63±0.13（5.3％），0.52±0.07（4.3％），1.14±0.18（9.5％），1.33±0.21（11.1％），2.76±0.16（23.0％）和 2.95±0.13mg/ml（24.5％），与使用 SBTI 作为外标获得的结果相似（P＞0.05）。比较采集方法，我们发现 Schirmer 试纸采样会导致泪液中带 2（主要是 HSA）的浓度增加，并且还会减少另外五个主要的泪液蛋白条带。比较不同年龄组之间的总蛋白浓度，我们发现年轻人的总蛋白浓度较高（P＜0.05）。这种高蛋白质水平是由所有主要的泪液蛋白成分共同贡献的。
使用 SBTI 作为内标，我们可以通过 SDS-PAGE 密度测定法从小量泪液样本中同时定量测定总泪液蛋白和主要泪液蛋白成分。该方法有望成为识别泪液蛋白变化的眼部疾病发生的诊断工具。
