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D. R. Powell, M. Thangavelu, J. J. Nichols; Comparison of Pooled and Non-Pooled Extracted Tear Protein Profiles From Silicone Hydrogel Lenses. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3409.
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Using pooled samples, which increases the volume or concentration of tear proteins available for analysis, is commonly done in the biochemical evaluation of tears. A concern with using pooled samples, however, is the potential to obscure the biological variability that may otherwise be deemed important. The purpose of this study was to evaluate protein differences in pooled and non-pooled contact lens samples from dry eye and non-dry eye soft contact lens wearers.
Dry eye status was classified by the Contact Lens Dry Eye Questionnaire. O2 Optix (lotrafilcon B, CIBA Vision) silicone hydrogel (SiHy) lenses were worn at least 12 hours daily for 14 days before collection by sterile tweezers and stored in glass vials at -86°C until analysis. To each sample, 1 mL 50:50 0.2% trifluoroacetic acid/acetonitrile was added and chilled at -20°C for 16 hours. The supernatant was evaporated and the pellet resuspended in 25 µL HPLC-grade water and reserved for Bradford assay. Equal protein concentrations were used for all individual samples and the pooled sample by determining the volume needed for 3 µg. Deionized water and 5 µL loading buffer was added, heated at 95°C for 5 minutes, then allowed to cool before 1D SDS-PAGE using 12% Tris-acrylamide gel. Gels were stained with silver and then imaged with the Kodak Image Station 4000MM (Kodak, Rochester, NY). Densitometry quantified three selected individual protein bands (80, 63, and 24 kDa) used in the analysis. One sample t-tests compared the differences in band intensities between all non-pooled samples to the pooled (population) sample.
Sixteen dry eye (29.9 ± 9.4 years, 81.3% female) and non-dry eye-classified contact lens wearers (26.2 ± 5.2 years, 87.5% female) participated in the study (n = 32). Protein concentration for the dry eye and non-dry eye-classified samples were 9.84 ± 8.92 and 12.09 ± 11.51 µg/lens, respectively. A significant difference in signal intensity was found in the 80 kDa and 24 kDa bands between the pooled and non-pooled samples for both the dry eye (p = 0.09 and p < 0.001) and non-dry eye-classified subjects (p = 0.02 and p = 0.03). No significant difference was found at 63 kDa between the pooled and the non-pooled samples for both groups (p = 0.22 for the dry eye and p = 0.45 for the non-dry eye group).
Pooled or non-pooled extracted tear proteins from SiHy lenses may offer the most optimal yield of available deposited protein depending on the protein band of interest. The results of this study concur with previous findings conducted in our lab from microcapillary-collected dry eye tear samples (80 kDa, p < 0.001; 63 kDa, p = 0.75; 24 kDa, p = 0.02).
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