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Kwaku Osei, Champion Deivanayagam, Jason J Nichols; Determination of DMBT1 immobilization on soft contact lens. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1750.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that when immobilized/adsorbed on a surface, Deleted in Malignant Brain Tumor-1 (DMBT1) and its Scavenger Receptor Cysteine-rich (SRCR) domains promote infections. To begin to understand the role of DMBT1 in contact lens-related infections, the immobilization of tear DMBT1 as well as the recombinant SRCR1 domain were investigated.
To determine the immobilization of SRCR on contact lens materials, three lenses each of etafilcon A and lotrafilcon B were soaked in 1 ml of purified SRCR1 solution at the following concentrations: 0.8, 0.2, 0.05, 0.0125, and 0 mg/ml (negative control) (n=15 per material). Lenses were incubated for 12 hours at 37 °C after which they were washed, blocked, and probed with anti-DMBT1 primary antibody and Alexa-488 conjugated IgG. Four different quadrants of the lens surface were imaged using a fluorescent microscope. Fluorescence from immobilized SRCR1 was analyzed using ImageJ, where both areas of SRCR1 immobilization and their relative intensities were determined. The area of SRCR1 immobilization was normalized by dividing the area of immobilization by the total surface area of the imaged region of the lens. To determine the adsorption of DMBT1 on worn lenses, protein adsorbed on 8 worn monthly-disposable lotrafilcon B lenses was extracted and pooled. Dot blot analysis was used to determine the adsorption of DMBT1.
SRCR1 was found to be immobilized on both contact lens materials. Across all concentrations, no significant difference was found in the normalized area of SRCR1 immobilization between etafilcon A and lotrafilcon B (Mann-Whitney U Test, p = 0.23). The relative immobilization intensity was higher for etafilcon A (224.706 vs 121.870, Mann-Whitney U Test, p = 0.02). The area of SRCR1 immobilization and intensity of immobilization were dose-dependent at higher concentrations of SRCR1 (0.8 – 0.05 mg/ml) for lotrafilcon A. Likewise, DMBT1 was detected in the pooled protein extract from worn lotrafilcon B lenses, an indication that DMBT1 is immobilized on worn lotrafilcon B lenses.
DMBT1 and SRCR1 are immobilized on etafilcon A and lotrafilcon B lenses. Further work is needed to determine if immobilization of DMBT1 plays a role in contact lens-related infections.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Immobilization of SRCR1 on lenses (A). DMBT1 was detected on dot blot analysis (B).
Area of SRCR1 immobilization (A) and Intensity of immobilization on lenses (B) determined with ImageJ.
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