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C. J. Giasson, A. Abouchita, S. Djouahra, V. Moore, R. Paquette; A Modified HPLC Technique to Measure Lysozyme Levels on Contact Lenses. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5395.
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To increase the sensitivity of a gradient elution reversed-phase HPLC method designed to analyze lysozyme levels contained within contact lens extracts.
Fifteen worn contact lenses incubated in a 50:50 solution of 0.2% trifluoroacetic acid (TFA): acetonitrile (ACN) for 20 hours or more in order to extract protein. Extracts were evaporated under vacuum and the remaining solids dissolved into a 0.1% TFA solution of 15% ACN:85% water (initial mobile phase) to obtain an enrichment factor of 4. Enriched extracts were separated by a C18 TSK NPR (4.6 x 35 mm) column of a HPLC system equipped with a UV-visible diode array detector set at 220 nm. Proteins were separated by gradient elution with 0.1% TFA in ACN (eluant A) and 0.1% TFA in water (eluant B). The initial gradient of 15% of eluant A ramped to reach 65% 6 minutes after the injection. Subsequent analysis was restricted to the 4 most concentrated extracts. Lysozyme contents of contact lens extracts were calibrated with the linear regression equation of lysozyme peak surface area as a function of known lysozyme content of injected standards. The collected fractions, concentrated by ultrafiltration, were analyzed with anti-human lysozyme in a western blot.
Two main peaks, observed at 3.5 ± 0.1 and 5.1 ± 0.1 minutes had a mean bandwidth at half-height of 8.5 ± 1.6, and 6.8 ± 1.2 seconds, respectively. Western blot analysis revealed that the fractions collected at 5 minutes contained lysozyme. The 4 extracts of 4 different contact lenses that had the highest levels of lysozyme ranged between 3.6 ± 0.3 and 6.9 ± 0.6 µg of lysozyme in the enriched extracts.
A step of enrichment of the protein extract coupled with solubilization into the initial mobile phase can be useful to improve sensitivity of this HPLC method in order to detect levels of lysozyme otherwise below the sensitivity of the measurement. Further increases in the enrichment ratio could be useful to decrease the limit of detection of this method.
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