Purpose: Silicone-hydrogel (SH) contact lens materials such as balafilcon (Purevision (PV), Bausch&Lomb) and lotrafilcon (Focus Night&Day (FND), CIBA) deposit significantly less protein compared to conventional hydrogel contact lens materials. Consequently, traditional total protein assays such as the Bradford and micro-BCA lack the required sensitivity to accurately quantify SH total protein deposits. The purpose of this work was to optimize a procedure to accurately and reproducibly quantify total protein in the low to mid nanogram range.

Methods: Four protein assays were assessed: (a) CBQCA (Molecular Probes); (b) red-dot-blot (RDB); amido black (AB) dot-blot onto (c) nitrocellulose (NC) and (d) polyvinylidene difluoride (PVDF) membranes. Where necessary, membranes were imaged and densitometry performed using a Syngene Gene Genius Ô Gel Documentation System and associated software. Assays were evaluated for 1) the lower limit of sensitivity, 2) accuracy [as compared to a standardized set of purified normal human tear film proteins individually quantified via Ninhydrin assay using BSA as standard], and 3) cross-reactivity toward unworn PV, FND and etafilcon (Acuvue (AV), Vistakon) contact lenses extracted with 1.5 ml of 50:50 acetonitrile:0.2% trifluoroacetic acid.

Results: The CBQCA assay provided the greatest sensitivity with a lower detection limit of 10 ng. Detection limits for both AB assays and the RDB were 25 and 100 ng, respectively. All protein assays except RDB accurately quantified hen egg lysozyme and human albumin. AB on PVDF exhibited the greatest quantification accuracy for human lysozyme, bovine lactoferrin and proteins with very low isoelectric points, as represented by human a1 acid glycoprotein (pI 2.7). AB on NC and the RDB most accurately quantified mucin from bovine submaxillary glands and pooled human tears. Cross reactivity to extracts of unworn SH contact lens was observed with all assays except AB on NC.

Conclusions: We conclude that our optimized AB on NC protein assay provides a reliable and sensitive means for quantification of total protein as low as 50 ng in protein extracts of SH contact lenses.