Stabilization of lysozyme mass extracted from lotrafilcon silicone hydrogel contact lenses.
OBJECTIVE
Lysozyme deposits extracted from lotrafilcon silicone hydrogel (SH) contact lens materials demonstrate a loss in total mass as a function of storage time when assessed by Western blotting. This loss represents a potential source of error when quantifying total lysozyme deposition on SH lenses. The purpose of this study was to devise a method whereby lysozyme mass would be preserved over time to allow for its accurate quantitation after its removal from SH lenses.


METHODS
Lysozyme deposits from 12 human worn lotrafilcon lenses were extracted using a 50:50 mixture of 0.2% trifluoroacetic acid and acetonitrile. Extracts were lyophilized to dryness, then resuspended in either reconstitution buffer (10 mM Tris-HCl, 1 mM EDTA) or modified reconstitution buffer (reconstitution buffer + 0.9% saline). BIOSTAB Biomolecule Storage Solution (Sigma-Aldrich) was added to one half of the samples from each buffer group. One microliter of each of the samples was immediately subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting, whereas the remaining volume was aliquoted and stored at -20 degrees C or -70 degrees C and subjected to the same procedures after 48 h of storage. Comparison of lysozyme band intensity in stored vs. fresh samples enabled calculation of percentage mass loss of lysozyme.


RESULTS
Samples stored at -20 degrees C in reconstitution buffer with no BIOSTAB demonstrated a 33% loss in mass over 48 h of storage. Identical samples stored at -70 degrees C in modified reconstitution buffer with BIOSTAB added demonstrated <1% loss in mass. Statistical analysis indicated that buffer composition (p < 0.001), storage temperature (p = 0.04), and addition of BIOSTAB (p < 0.001) were all important in controlling loss of mass over time.


CONCLUSIONS
We have optimized a procedure whereby the extracted mass of lysozyme deposits found on lotrafilcon SH lenses can be preserved, thus enabling accurate quantitation after extraction and resuspension.
Subbaraman
Lakshman N
LN
Centre for Contact Lens Research, School of Optometry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Glasier
Mary-Ann
MA
Senchyna
Michelle
M
Jones
Lyndon
L
D015153:Blotting, Western; D018639:Contact Lens Solutions / Q000737:chemistry; D003263:Contact Lenses, Hydrophilic; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D020100:Hydrogels / Q000737:chemistry*; D066298:In Vitro Techniques; D009113:Muramidase / Q000032:analysis / Q000302:isolation & purification*; D012828:Silicones / Q000737:chemistry*; D005466:Fluorocarbons
D018639:Contact Lens Solutions; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D020100:Hydrogels; D009113:Muramidase; D012828:Silicones; C481129:lotrafilcon A; D005466:Fluorocarbons