Impact of collection methods and storage conditions on the recovery of tear proteins

Abstract

AIM: To explore the optimal ways for the collection and preservation of tear proteins. 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 BCATM 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.

Keywords

• tear protein
• Shirmer's tear strip
• microcapillary tube
• microcapillary tube