A protein dye-binding assay on cellulose membranes for tear protein quantification: use of conventional schirmer strips.

PURPOSE: To develop a method to quantify tear protein concentration with the sensitivity to measure this variable in the restricted volumes of single human tear samples. METHODS: Aliquots of tear fluid from healthy subjects and a solution of standard bovine serum albumin (BSA) were spotted on cellulose membranes. Membranes were fixed, stained for protein with Coomassie blue, and washed until they displayed clear backgrounds. Stained spots were excised and eluted in a defined volume of methanol-ammonia, and the absorbance was determined spectrophotometrically at 610 nm. Membranes were calibrated by calculating their apparent thickness from the areas of stained spots and the corresponding aliquot volumes of either tear fluid or BSA solution. RESULTS: In our dye-binding assay, absorbance (0-1.00 OD) was found to have a linear relation with tear fluid volume (1-7 microL). In a study involving samples from 33 healthy subjects, aliquots (3 microL) of tear fluid were found to yield absorbances in the linear range. Protein concentrations in tear fluid were found to be distributed over the range of 2.20-6.37 mg/mL (mean, 4.11 +/- 1.00 mg/mL) with no apparent sex differences. The assay can be applied successfully to quantify protein concentrations in tear fluid by using calibrated Schirmer strips after a tear test. Electrophoretic profiles of proteins present in tear fluid sampled from different healthy individuals were nearly identical when normalized for protein load by using this method. CONCLUSIONS: The protein dye-binding assay we developed by using cellulose membranes or Schirmer strips is an efficient and convenient method for measuring tear protein concentration.

Use of polyurethane minisponges to collect human tear fluid.

PURPOSE: To characterize a method of tear collection based on the use of amphiphilic polyurethane absorbing minisponges. METHODS: Tear fluid was collected from 17 healthy volunteers. A preweighed polyurethane dry minisponge was laid on the margin of the lower eyelid. Once wet (5-10 minutes), the fluid was transferred to a preweighed Eppendorf tube after squeezing the sponge by centrifugation. The amount of fluid absorbed and fluid recovered were determined by reweighing the sponge and the tube after absorption and centrifugation steps, respectively. The fluid was qualitatively characterized by electrophoretic polypeptide profiling in Coomassie blue-stained SDS-polyacrylamide gels. RESULTS: Per eye, 14.6 +/- 5.3 microL tear fluid was collected. That volume was about 90% of the fluid absorbed by polyurethane minisponges, almost doubling the fraction recovered from other more hydrophilic absorbing polymers. Major bands characterizing the electrophoretic profile of this fluid were those of 79, 66, 27, 18, and 14 kd. This profile was indistinguishable from that of tear fluid aspirated into glass microcapillaries. Tear fluid collected simultaneously from both eyes displayed the same profiles. Successive tear samples from a single eye showed the same profile except for the 66-kd band, which increased steadily as collection proceeded. Tear donors rarely complained of discomfort. CONCLUSIONS: Tear collection by absorbing polyurethane minisponges is highly advantageous in efficiency (recovery) and reproducibility (invariant electrophoretic polypeptide profiles). Tear donor comfort, simultaneous bilateral collection, and collections from several donors at once are additional major advantages of this collection method in studies involving single subjects and populations in health and disease.