Purpose: Preliminary results in our laboratory demonstrated that lysozyme deposits extracted from silicone-hydrogel (SH) contact lens materials demonstrated a loss in total mass as a function of storage time when assessed by Western blotting (WB). This loss represents a potential source of error when quantifying total lysozyme deposition. Thus, the purpose of this work was to devise a method whereby lysozyme mass would be preserved over time and would be compatible with our previously described WB procedure (1).
Methods: Lysozyme deposits from human worn 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 (RB) (10mM Tris-HCl, 1mM EDTA) or Modified Reconstitution Buffer (MRB) (RB + 0.9% saline). BioStab (1 in 4 parts) (Sigma) was added to one half of the samples from each buffer group. 1 mL of each of the samples was immediately subjected to SDS PAGE and WB on a PhastSystem (Pharmacia), while the remaining volume was aliquoted and stored at -20 °C or -70 °C and subjected to the same procedures after 48 hours of storage. All WBs were imaged and quantified on a Storm 840 Imaging system. Comparison of lysozyme band intensity in stored versus fresh samples enabled calculation of percentage mass loss of lysozyme.
Results: As can be seen in Table 1,resuspension of lyophilized SH lens extracts in MRB and BioStab provided essentially 100% protection to lysozyme mass when stored in frozen aliquots.
Conclusion: We have optimized a procedure using MRB, BioStab and storage at -70 oC, whereby the extracted mass of lysozyme deposits found on SH lenses can be preserved without loss to facilitate accurate quantitation via our WB procedure .
Table 1: Percentage mass loss of Lysozyme after 48 hours of storage:
|
-20 ° C |
P |
-70 ° C |
P |
No Biostab |
With Biostab |
No Biostab |
With Biostab |
RB |
33.2% ± 0.5 |
15.8% ±0.5 |
<0.001 |
31.3% ±1.5 |
14.1% ±0.9 |
<0.001 |
MRB |
19.2% ± 4.5 |
0.9% ± 0.8 |
<0.001 |
17.1% ± 2.8 |
0.6% ± 0.8 |
<0.001 |
P |
<0.001 |
<0.001 |
|
<0.001 |
<0.001 |
|
1. Senchyna M, Jones L, Louie D, May C, Forbes I, Glasier M: Quantitative and conformational characterization of lysozyme deposited on balafilcon and etafilcon contact lens materials. Curr Eye Res 2004; 28: 25-36.
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