Tear Protein Concentration Research Articles

A highly sensitive chemiluminescence immunoassay for the measurement of coagulation factor XIII subunits and their complex in tears

Blood coagulation factor XIII (FXIII) is a tetramer (A 2B 2) consisting of catalytic FXIII-A and carrier/protective FXIII-B subunits. Besides stabilizing fibrin and protecting it from fibrinolysis, FXIII is also involved in wound healing and angiogenesis. The latter functions could be important in body fluids other than blood. In this study highly sensitive chemiluminescent ELISAs were developed to measure low concentrations of FXIII-A, FXIII-B and FXIII-A 2B 2 antigen levels in tiny volumes of tear. For all three assays the limit of quantitation was below 0.02 µg/L, within laboratory imprecision did not exceed 12% and the recovery was close to 100%. The calibration curves showed an excellent fitting using second order polynomial equation. In non-stimulated tears from 40 healthy volunteers a low, but consistent amount of FXIII-A and FXIII-B (medians: 1.74 µg/L and 5.73 µg/L, respectively) was measured, mostly in free non-complexed form. The respective median values normalized for tear protein concentration were 0.23 µg FXIII-A/g and 0.85 µg FXIII-B/g. There was no gender and age difference and the values showed non-Gaussian distribution. The results prove that FXIII-A and FXIII-B are low concentration components of tear proteome, which in normal conditions might play a role in the repair of corneal micro-injuries. The developed methods provide a tool for monitoring FXIII subunit and complex levels in pathological conditions.

Tear sample collection using cellulose acetate absorbent filters

To assess the use of cellulose acetate filter rods as a technique for tear collection. The cellulose acetate rod (CR) was compared with the 'standard' glass capillary tube (CT), in a series of experiments, to assess: sample collection by collected volume size; the effect of tear stimulation on total tear protein concentration and major tear protein concentrations; and technique invasiveness. No difference was found in concentrations for total protein, IgA (secretory immunoglobulin A), lactoferrin and lysozyme (p > 0.05) with no clinically significant increase in serum albumin to show serum leakage. Sample volume was higher for CR (p < 0.005) and sample volume increased for stimulated collection with CR (p = 0.001). Dilution effect of a stimulated sample size was reliably shown only with CR (r = -0.66, p = 0.011). Using bovine albumin standard with CR and CT, a smaller sample volume (p < 0.001) and a higher protein concentration (p < 0.001) were extracted with CR. The cellulose rod offers a suitable alternative to the glass CT. It is able to quickly absorb a sample, allowing use for a wide range of sample sizes, while being minimally invasive.

Analysis of human tear fluid by Raman spectroscopy

Tear fluid is a complex aqueous solution containing proteins, metabolites, electrolytes and lipids. This study uses Raman spectroscopy to analyse the composition of human tear fluid from three healthy volunteers. Two different methods are used to obtain Raman spectra from the 3 μL tear samples: (i) solution-phase Raman spectroscopy, and (ii) drop coating deposition Raman spectroscopy (DCDRS). Tear samples were either basal fluid, or yawn reflex secreted fluid. Calibration of the solution technique with standard protein solutions (5–15 mg mL −1) showed that this method could predict the protein concentration (cross-validation) with an error of less than 1 mg mL −1. The Raman signals from the tear fluid were very weak but signals due to protein and urea were clearly observable in all samples. The drop coating deposition technique was shown to produce very high signal-to-noise spectra for relatively short acquisition times, and small sample volumes. Raman point mapping combined with principal components analysis showed that the protein, urea, bicarbonate and lipid could all be detected in the tear samples and that the distribution of these components was inhomogeneous. Their position within the drying pattern was shown to depend on their relative solubilities. The results of this study suggest that solution Raman measurements may be calibrated to give the total tear protein concentration and DCDRS could be used to give a fingerprint of the tear protein (and lipid) composition.

Lacritin, a Novel Tear Glycoprotein, Stimulates Tear Production in Rabbits without Ocular Toxicity

Dry eye is extremely common and debilitating disease. Current treatment options like artificial tears and cyclosporin are palliative and do not effectively address the underlying pathology of dry eye. Lacritin is a newly discovered glycoprotein that occurs naturally in human tears, produced solely by meibomian, lacrimal and salivary glands. Human recombinant lacritin stimulates tear secretion in cultured rat lacrimal acinar cells, leading us to hypothesize that topical application of lacritin will increase production of tears of normal composition. Bilateral tear production in New Zealand white rabbits was measured at 60, 120 and 240 min after a single topical administration of 50μl lacritin (1, 10, or 50 μg/ml). Tears were collected and slit lamp examination and ocular surface staining measured before and 240 min after treatment. Tear pH, protein and electrolyte concentrations were then measured. A 50 μg/ml lacritin dose increased tear production by 40% within 60 min and maintained for 240 min (p&lt;0.05). With other doses, a significant increase (55% from baseline, p&lt;0.05) was observed only at 240 min. No local irritant effects were noted by slit‐lamp examination. The composition (pH, Na2+ and K+) of tears was also not different from normal tears. In conclusion, lacritin is a novel, safe and effective candidate for dry eye therapy. Lacritin, being mitogenic, may also help restore the natural tear film.

A Protein Dye-Binding Assay on Cellulose Membranes for Tear Protein Quantification

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. 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. 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. 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.

Topical 0.1% Prednisolone Lowers Nerve Growth Factor Expression in Keratoconjunctivitis Sicca Patients

To compare nerve growth factor (NGF) levels in tears and on the ocular surfaces of normal control and non-Sjögren's type keratoconjunctivitis sicca subjects, and to investigate the effect of 0.1% prednisolone eyedrops on NGF levels in keratoconjunctivitis sicca patients. Prospective, double-masked, randomized, comparative clinical trial. Forty-one keratoconjunctivitis sicca patients and 23 age- and gender-matched healthy subjects. Baseline tear NGF levels were measured in keratoconjunctivitis sicca patients and healthy control subjects using enzyme-linked immunosorbent assays. Keratoconjunctivitis sicca patients received 0.1% prednisolone drops in one eye and 0.1% hyaluronic acid drops in the other, 3 times a day for 28 days. Also, impression cytology (IC) and immunostaining for NGF on conjunctival epithelium were performed on both groups. Tear NGF/total tear protein (TP) concentration ratio, IC and NGF immunocytologic staining, subjective symptom scale, tear breakup time, and Schirmer values. Keratoconjunctivitis sicca patients were found to have baseline tear NGF concentrations higher than those of age- and gender-matched healthy control subjects (65.9+/-14.5 vs. 122.1+/-45.3 pg/mug, P<0.0001). In keratoconjunctivitis sicca patients, prednisolone treatment for 28 days resulted in a decrease in tear NGF levels, symptom scores, and IC scores, whereas hyaluronic acid treatment had no such effect (68.2+/-25.0 pg/mug vs. 108.0+/-43.4 pg/mug, P<0.0001 for tear NGF/TP ratio; 2.16+/-1.01 vs. 3.39+/-1.50, P = 0.0014 for symptom scale; 1.05+/-0.67 vs. 1.61+/-0.86, P = 0.0317 for IC). Measurements taken at both 14 and 28 days indicated that neither prednisolone nor hyaluronic acid treatment affected breakup time or Schirmer values. Keratoconjunctivitis sicca patients showed elevated levels of tear NGF, which were decreased by treatment with 0.1% prednisolone. These data suggest that ocular surface NGF may play an important role in ocular surface inflammation processes associated with dry eyes.

Temporal Changes in Tear Proteins During Onset of Dry Eye Disease in a Mouse Model of Sjögren's Syndrome

Purpose. The NOD mouse, as well as the newly constructed C57BL/6.NOD-Aec1Aec2 mouse, have been shown to exhibit numerous disease parameters that parallel Sjogren’s syndrome (SjS) in humans, including loss of stimulated fluid secretion concomitant with the appearance of leukocytic infiltrates in the lacrimal and salivary glands. In the present study, we have catalogued the temporal changes in tear proteins with the onset of dry eye disease in these two mouse strains. Methods. Tears, collected from CD-1, NOD.B10-H2b and C57BL/6.NOD-Aec1Aec2 mice at 3, 8, 14, and 20 weeks of age, were labeled with either Cy-3 (green) or Cy-5 (red) dye. Labeled tear proteins were separated individually on pH3-10 NL18 cm IPG strips, then electrophoresed through 8-16% Tris-HCl SDS-polyacrylamide gels. Each gel was scanned for visualization using ImageQuant software. Each gel was compared by ImageMaster 2-D Elite software to identify unique protein difference. Each protein spot was sequenced using MOLDI-TOF. Results. NOD.B10-H2b and C57BL/6.NODAec1Aec2 mice exhibited marked losses in their tear protein concentrations compared to CD-1 mice. To date, nearly 50 protein spots have been compared between the autoimmune and nonautoimmune mice. Some 10 tear proteins remained unchanged through 20 weeks of age (e.g., lacrimal androgen binding protein); however, the majority of tear proteins showed some increased levels in the autoimmune mice (e.g., DNAseI orglobulin IV precursor), while >12 spots were lost in conjunction with the onset of dry eye disease. Conclusions. Onset of dry eye disease in mice exhibiting SjS-like disease is associated with loss of tear volume, decreased tear protein concentrations, loss in expression of several tear proteins, plus appearance of altered proteins. Commercial Relationship(s): ABP is a scientific consultant for and stockholder in Ixion Biotechnology, Inc.; Support: NIH grants DE13769, DE014344 & DE015152

Tear proteins of normal young Hong Kong Chinese

Analysis of tear proteins is of diagnostic value for abnormal ocular conditions such as dry eye syndrome. Many studies of tear proteins have been performed on Caucasian subjects. However, little is known about these proteins in Chinese eyes. The total tear protein concentrations of 30 normal young Hong Kong Chinese were determined by the Bradford method and the modified Lowry method. Bovine serum albumin (BSA) and bovine immunoglobulin G (IgG) were both used as standards for each method. The tear protein patterns were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentrations of major tear proteins were quantified by scanning densitometry after SDS-PAGE. The mean +/- SD total tear protein concentrations determined by the Bradford method, using BSA and IgG as standards, were 6.05 +/- 1.58 mg/ml and 11.48 +/- 2.32 mg/ml respectively. The values determined by the modified Lowry method, using the same two standards, were 9.66 +/- 2.03 mg/ml and 7.53 +/- 1.80 mg/ml respectively. The mean +/- SD concentrations of major tear proteins were 2.73 +/- 0.82 mg/ml for lactoferrin, 0.021 +/- 0.028 mg/ml for human serum albumin, 2.89 +/- 0.88 mg/ml for tear-specific prealbumin and 2.46 +/- 0.44 mg/ml for lysozyme. The results of total tear protein concentrations indicated that values obtained from different methods and different standards were not comparable. The tear protein patterns of our subjects were qualitatively similar to those reported for Caucasian subjects. However, the concentrations of the major proteins of our subjects were not in accordance with those reported previously. The main reason may be the large variability of method used.

Denervation of rabbit lacrimal gland increases levels of transferrin and unidentified tear proteins of 44 and 36 kDa

Purpose. The main lacrimal gland secretes proteins and fluid that make up the aqueous component of the tears. Previous reports indicate that the parasympathetic innervation of the gland influences the secretion of protein from the lacrimal gland. We investigated the effect of lacrimal nerve transection on the levels of individual proteins and overall protein concentration in the tear fluid. Methods. The main lacrimal gland was unilaterally denervated in adult rabbits at the site of nerve entry to the gland. The contralateral gland (sham-operated) had identical surgical manipulations, excluding nerve transection. Tears were collected daily from both eyes for up to 9 days, after which lacrimal glands were collected. SDS-PAGE, densitometric and image analysis, and Western blot were performed. Results. Consistently measurable tear protein bands ranged from 6 kDa to 85 kDa, using densitometric analysis. Lacrimal gland denervation produced a sustained increase in proteins of 85, 44, and 36 kDa in tears and lacrimal gland tissue from the denervated side, compared with the sham-operated side (0.025 > p > 0.001). The band at 85 kDa was identified as transferrin by Western blot. Tears from the denervated glands also showed transient decreases in low molecular weight tear proteins (18, 12/10, and 6 kDa), as well as a decrease in overall protein concentration, compared with tears from sham-operated glands and non-operated glands (p < 0.001). Conclusions. These results demonstrate that, in rabbit tears, the quantities of transferrin and two unidentified tear proteins, as well as overall protein concentration, are influenced by the sensory and/or autonomic innervation to the lacrimal gland. The decrease in overall tear protein concentration after lacrimal gland denervation may be related to a loss of nerve-regulated secretagogue-induced protein secretion.

Tear changes in contact lens wearers following overnight eye closure

PURPOSE. Tear protein composition alters during eye closure, by becoming rich in secretory IgA (sIgA) and certain complement proteins. This may reflect altered ocular defense mechanisms during eye closure. Since overnight wear of contact lenses (CLs) is associated with an increased risk of corneal infection and inflammation, this study aimed to quantify tear protein changes with overnight soft CL wear. METHODS. Non-stimulated tears were collected from 9 CL wearers prior to CL wear (baseline), after daily CL wear, and after 8 h sleep. Lenses were removed following wear and were extracted in 80% urea at 95°C. Secretory IgA, complement C3 and C4, were measured using ELISA and total protein using the Pierce BCA assay. Assays were performed on tear samples and CL extracts. RESULTS. Baseline tear protein concentrations were: 0 total tear protein (9.37 ± 2.97 mg/mL), C3 (4.4 ± 2.1 µg/mL), C4 (0.1 ± 0.1 µg/mL), and sIgA (0.84 ± 0.34 mg/mL). There were no differences in any protein levels between daily CL wear and no CL wear (p > 0.05). Following sleep, protein concentrations were: total tear protein (43.64 ± 24.30 mg/mL), C3 (72.5 ± 49.9 µg/mL), C4 (6.7 ± 5.2 µg/mL), and sIgA (5.53 ± 5.15 mg/mL). Total protein extracted from CLs after daily wear was 90 ± 27 µg/CL and, after overnight wear, 152 ± 24 µg/CL. Negligible levels of C3, C4 and sIgA were recovered from CL extracts. CONCLUSIONS. Uncomplicated daily use of soft CLs does not appear to alter certain tear proteins compared with baseline levels. Uptake of these proteins by the CL does not appear to deplete the tear protein levels. Overnight levels of all tear proteins were increased, compared to daily CL wear.

Immunoglobulins in stimulated tears of dogs

Summary Immunoelectrophoresis and single radial immunodiffusion were used to identify and measure tear immunoglobulin concentrations in 50 healthy dogs. Immunoglobulin A and IgG were detected in all samples analyzed, whereas IgM was not detected in any sample. Mean IgA concentration was 25.28 ± 1.9 mg/dl, adult dogs (&gt; 18 months) having significantly higher mean value. The IgA concentration related to age had significant (P &lt; 0.006) positive correlation; mean IgG concentration was 23.10 ± 1.72 mg/dl. Linear correlation analysis revealed significant (P &lt; 0.0007) correlation coefficient between tear total protein and IgA concentrations. The IgA and IgG concentrations also were significantly (P &lt; 0.0001) correlated when expressed as milligrams per 100 mg of protein. Relation with sex was not established for either immunoglobulin.

A human model of allergic conjunctivitis.

Using the method of refractometry to measure protein concentration in tears, a simple model was developed to evaluate the allergic response in humans. Timothy grass pollen was instilled into the cul de sac of a human subject, and the protein content of the subject's tears was sampled every 15 minutes for three hours. Once the time course of this experiment was predictable, various eye medications were instilled into the subject's eye 30 minutes after allergic challenge. It was noted that 1.0% and 0.1% prednisolone, each combined with 0.12% phenylephrine hydrochloride, as well as 1% epinephryl borate eye drops, produced an almost immediate return to normal of tear protein levels, whereas 1.0% medrysone, 0.1% fluorometholone, 1.0% prednisolone, and 0.12% prednisolone had less pronounced effects on reduction of elevated tear protein concentration.