Optimization of a Fluorescence-based Lysozyme Activity Assay for Contact Lens Studies
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PURPOSE: To optimize a fluorescence-based lysozyme activity assay to investigate the conformational state of lysozyme in solution and to determine the impact of extraction and evaporation procedures and the possible interference of contact lens materials on lysozyme activity. METHODS: The fluorescence-based lysozyme activity assay, Enzchek (Molecular Probes Inc, Eugene, OR) which utilizes fluorescently quenched Micrococcus lysodeikticus, was compared to the gold standard, classical lysozyme turbidity assay, using four differently concentrated lysozyme samples (20, 10, 5.0 and 2.0 ng/µL). Furthermore, six differently concentrated lysozyme samples (2.0, 1.0, 0.5, 0.25, 0.125 and 0.01 µg/µL) were quantified using the fluorescence-based assay in the presence of extraction solvents consisting of 0.2% and 0.02% trifluroacetic acid/acetonitrile and following evaporation procedures. RESULTS: A standard curve was generated by the fluorescence-based assay ranging from 2 to 150 ng. The total active lysozyme quantified in the four lysozyme samples was not significantly different between the two assays (p > 0.05) and the concordance correlation coefficient was determined to be 0.995. However an average discrepancy between the two assays was found to be 0.474 ng, with the turbidity assay typically reporting higher active lysozyme measurements. The sensitivity of the fluorescence-based assay was higher than the classical turbidity assay when quantifying 20 ng or less active lysozyme. Following the extraction and evaporation procedures and the addition of lens extracts, the total active lysozyme recovered was 95% or greater. CONCLUSIONS: In comparison to the classical turbidity assay, the fluorescence-based assay is a very sensitive method, making it a favorable technique, particularly when studying contact lens materials that deposit relatively low levels of lysozyme.
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