A Solid-Phase Assay for the Quantitation of Total Protein Eluted from Balafilcon, Lotrafilcon, and Etafilcon Contact Lenses
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abstract
PURPOSE: To compare two variations of a membrane-based protein assay utilizing Amido black (AB) detection with a commercially available 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) assay for use in the quantitation of individual tear proteins, pooled human tear proteins, and protein extracted from ex vivo lotrafilcon A, balafilcon A, and etafilcon A contact lens materials. METHODS: Ex vivo contact lens extracts, pooled human tears, and individual tear proteins (human serum albumin (HSA), bovine lactoferrin, human secretory immunoglobulin A (sIgA), human lysozyme) were subjected to three solid-phase assays: AB on polyvinylidene difluoride (AB on PVDF) and AB on nitrocellulose (AB on NC) and the CBQCA assay. Micro-bicinchonic acid (micro-BCA) assay was also employed with lens extracts to determine total protein concentration. Individual and pooled tear proteins were referenced to a micro version of the quantitative ninhydrin protein assay. RESULTS: The CBQCA demonstrated the greatest overall sensitivity and lowest intra- and inter-assay variability. AB on NC demonstrated the most accurate ability to quantify total protein in pooled human tear samples, although it also displayed the greatest protein-to-protein variation using individual tear proteins. The CBQCA assay displayed the greatest cross-reactivity with unworn balafilcon and lotrafilcon lens extracts, whereas AB on NC demonstrated the least. AB on NC measured similar amounts of total protein in extracted ex vivo lenses as the CBQCA assay if background interference was subtracted from CBQCA values. AB on PVDF measured the lowest amount of deposited protein from ex vivo lenses. CONCLUSION: Both the AB on NC and CBQCA assays can be used to measure protein in extracts of lotrafilcon, balafilcon, and etafilcon lens materials.
