目的：比较三种泪液采集载体(进口吸附滤纸、国产吸附滤纸和泪液采集毛细管)在不同储存温度条件下对泪液蛋白保存的影响，为科学地选择泪液采集方式、准确测定泪液中的不同类型的蛋白分子提供实验依据。

方法：表面硅化处理的玻璃毛细管，国产泪液试纸及进口泪液试纸吸附等体积等量的血清白蛋白、胎牛蛋白或已知浓度的IL-1Ra、IL-6、MIG、IP-10、MMP-9、TIMP1、VEGF和HBD1标准品混合液后放于无菌离心管并分别保存于4℃、-20℃和-80℃。以适当的洗脱液进行洗脱后，用Micro BCA^TM和酶联免疫吸附法分别检测总蛋白浓度和各种特异性蛋白浓度。

结果：洗脱缓冲液中是否添加表面活性剂或蛋白酶抑制剂对滤纸载体的蛋白洗脱率没有显著影响。-20℃和-80℃保存条件下，三种泪液采集载体对样本总蛋白的平均洗脱率没有显著性差异。IL-6在三种泪液采集载体中的平均洗脱率均较高，且载体之间及不同保存温度环境之间均没有显著的差异。滤纸和毛细管载体对IL-1Ra和HBD1的滞留效应差异较大：IL-1Ra在滤纸和毛细管载体中的平均洗脱率分别为19%±15%和98%±6%； HBD1在滤纸和毛细管载体中的平均洗脱率分别为93%±8%和61%±3%。MIG在毛细管收集载体中的平均洗脱率显著高于滤纸载体，分别为87%±4%和69%±4%。毛细管载体对MMP-9和TIMP1的平均洗脱率分别为90%±5%和103%±7%，同样显著高于滤纸(分别为62%±3%和87%±5%)。样本载体保存于-80℃ 环境时，其洗脱液中VEGF和IP-10的平均洗脱率分别为82%±5%和72%±8%，明显高于其他温度环境保存的样本载体。

结论：检测泪液中不同目的蛋白时，需综合考虑采集方法和保存温度条件，选择最经济可靠的方法。用毛细管采集泪液适合大多数的蛋白定量检测，检测IL-1Ra、IP-10、MIG、MMP-9和TIMP1的泪液适合用毛细管进行采集。但是对于有明显极性的蛋白，例如HBD1，则要事先试验最有效的收集和保存方法。

METHODS: Siliconized microcapillary tube or Schirmer's strips made in China or in the USA were tested. All three carriers were loaded with equal volume and quantity of bull serum albumin(BSA), fetal bovine serum(FBS)or known concentrations of IL1-Ra, IL-6, MIG, IP-10, MMP9, TIMP1, VEGF and HBD1 mixture and kept at 4℃, -20℃ or -80℃. After a desired time of storage, the proteins were eluted with different elution buffer. Micro BCA^TM protein quantitative kit and enzyme-linked immunosorbent assay kits were used to determine total protein concentrations and the concentrations of the above biological molecules, respectively.

RESULTS: Neither protease inhibitors nor detergent were necessary for protein elution from the Schirmer's strip. When stored at -20℃ and -80℃, three tear carriers showed no differences in the recovery of total protein. The recovery of IL-6 from three carriers under different temperature was similar but the higher than other proteins. The recovery rate of IL-1Ra was 98%±6% from the microcapillary tube and 19%±15% from Schirmer's strip. The recovery rate of HBD1 was 93%±8% from the microcapillary tube and 61%±3% from Schirmer's strip. The recovery rate of MIG was 87%±4% from the microcapillary tube and 69%±4% from Schirmer's strip. Microcapillary tube also had less retention for MMP-9(90%±5%)and TIMP1(103%±7%)compared to Schirmer's strip(62%±3% vs 87%±5%). The strips made in China and in the USA showed similar retention to most of the proteins tested here. When stored at -80℃, the recovery rate for VEGF and IP-10 were 82%±5% and 72%±8%, respectively, significantly higher than that when stored at higher temperature.

CONCLUSION: The selection of tear sample carrier is dependent on target proteins to be recovered. Low temperature deep freezing is a preferred way for tear sample storage. microcapillary tube is applicable for most of the proteins. While for those protein with noticeable polarity, like HBD1, pre-experiment is needed.