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Brad Hall, Lyndon Jones, James A. Forrest; Measuring The Kinetics and Activity of Adsorbed Proteins: In Vitro Lysozyme Deposited Onto Contact Lenses Over Short Time Periods. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6125.
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To develop a process to measure the biological activity of an intact layer of adsorbed lysozyme at the surface of hydrogel contact lens materials.
We use this technique to measure the time dependent amount and activity of adsorbed lysozyme on a number of commercial contact lens biomaterials during the first 2 hours of protein interaction with the material surface. The quantity of adsorbed lysozyme is measured using standard radiolabeled protein. The activity of the surface adsorbed protein is measured using a standard micrococcal activity assay, with extra steps to distinguish between protein on the surface and protein in solution. We use the measured quantities in our experiment to estimate a total layer activity.
The amount of active protein is essentially independent of the total protein, and is similar to what one would expect from monolayer coverage. We calculate the amount for a theoretical monolayer of lysozyme on a contact lens to be 692-1035ng, depending on the orientation of lysozyme, and thus can calculate a percentage of active lysozyme in a surface layer for each lens type. The percent of active lysozyme for senofilcon A, lotrafilcon B, and comfilcon A were 3-4, 4-5, and 2-3 percent respectively. Balafilcon A had the highest percent activity in a surface layer for all the silicone hydrogels tested at 30-45 percent. Etafilcon A was the only conventional hydrogel tested and showed 78-117 percent activity in a surface layer of lysozyme, which was the highest of all lens materials tested.
This study has established an effective technique to evaluate the activity of an intact lysozyme coating on a biomaterial. Our results show that protein can rapidly deposit and can rapidly lose its biological function on biomaterials in as little as 10 seconds of protein-surface interaction. Despite the simplicity of the technique, to the best of our knowledge this is the first report quantifying the biological activity of an intact layer of surface-adsorbed protein on hydrogel materials.
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