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Brad Hall, Chau-Minh Phan, Lakshman Subbaraman, Lyndon Jones, James Forrest; Direct comparison between in situ versus extraction techniques for measuring adsorbed proteins: application to lysozyme deposited onto hydrogel contact lenses. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5467.
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© ARVO (1962-2015); The Authors (2016-present)
To compare two techniques for measuring the activity of lysozyme deposited onto hydrogel contact lens and to image the binding of Micrococcus lysodeikticus to contact lenses.
Using a previously described protein extraction technique and a recently developed in-situ technique, we measured the time dependent activity of adsorbed lysozyme on six different contact lens materials during the first minute and up to one week of interaction with the material surface. Total activity of extracted lysozyme, total in-situ activity and the activity of the outer surface layer of sorbed lysozyme were determined using the two different techniques. Micrococcal cellular interaction with surface-adsorbed lysozyme was imaged using confocal microscopy.
The differences between total extracted activities, total in-situ activities and surface activities were both measurable and material specific. In most cases the total extracted activity > total in-situ activity > surface activity. After one week, etafilcon A had the highest extracted activity at 137μg/lens, followed by omafilcon A, balafilcon A, comfilcon A, senofilcon A, and lotrafilcon B at 27.4μg, 2.9μg, 2.0μg, 0.5μg, and 0.3μg respectively. Subsequent removal of adhered micrococcal cells was greatest on balafilcon A, which had the highest surface activity, and lowest on lotrafilcon B, which had the lowest surface activity.
This study measured and made direct comparisons between two established techniques for measuring the activity of adsorbed lysozyme. Both techniques demonstrate material dependent differences in activity. While individual extraction activities demonstrate the activity of underlying layers of lysozyme or lysozyme within the matrix of the material, in-situ measurements allow conclusions to be drawn about only the biologically relevant lysozyme, and surface activity measurements reveal the activity of just the outer surface of lysozyme.
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