title: Extraction versus in situ techniques for measuring surface-adsorbed lysozyme.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 1 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.
  
  
  RESULTS
  The differences between total extracted activities, total in situ activities, and surface activities were both measurable and material specific. In most cases, total extracted activity is greater than total in situ activity, which, in turn, is greater than surface activity. After 1 week, etafilcon A had the highest extracted activity at 137 μg/lens, followed by omafilcon A, balafilcon A, comfilcon A, senofilcon A, and lotrafilcon B at 27.4, 2.85, 2.02, 0.46, and 0.27 μg/lens, respectively. Micrococcal cell adhesion was greatest on contact lenses with high contact angles, such as balafilcon A, omafilcon A, and senofilcon A and lowest on contact lenses with low contact angles, such as etafilcon A, comfilcon A, and lotrafilcon B. Subsequent removal/prevention 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.
  
  
  CONCLUSIONS
  This study has measured and made direct comparisons between two established techniques for measuring the activity of adsorbed lysozyme. The extraction technique determines the activity of underlying layers of lysozyme or lysozyme within the matrix of the material. Conversely, the in situ technique allows conclusions to be drawn about only the biologically relevant lysozyme including the activity of just the outer surface of adsorbed lysozyme.
authors.affiliation: *BSc †MSc ‡PhD, BSOptom, FAAO §PhD, FCOptom, FAAO ∥PhD Centre for Contact Lens Research, School of Optometry and Vision Science (all authors), Department of Physics and Astronomy and Guelph-Waterloo Physics Institute (LWJ, JF), University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Brad
authors.initials: B
authors.lastname: Hall
authors.forename: Chau-Minh
authors.initials: CM
authors.lastname: Phan
authors.forename: Lakshman
authors.initials: L
authors.lastname: Subbaraman
authors.forename: Lyndon W
authors.initials: LW
authors.lastname: Jones
authors.forename: James
authors.initials: J
authors.lastname: Forrest
mesh_terms: D000327:Adsorption; D001422:Bacterial Adhesion; D003263:Contact Lenses, Hydrophilic; D057075:Enzyme Assays; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry; D008837:Micrococcus / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D009113:Muramidase / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding
chemical_list: D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D009113:Muramidase