Human Tear Proteins Research Articles

Anonic Silicon Hydrogels Affect the Concentration of Proteins in Tears during Wear

Purpose The objective of this study was to quantitatively assess the concentration of human tear proteins in patients wearing contact lenses of various ionicities and determine whether differences were related to the incidence of corneal infiltrative events (CIE). Methods 24 subjects (samples) were randomly selected for spectral count analysis to obtain protein concentrations using LCMS analysis. The subjects were neophyte and ametropic with ages between 18 and 40; 6 wore control lenses, 8 wore TestLens1, and 10 wore TestLens2. 16 subjects experienced CIEs during the study. Results A pairwise multiple hypothesis test identified 7 proteins that significantly differed in concentration between TestLens1 and control, and 11 proteins that differed between TestLens2 and control. Of the 12 unique proteins, 9 were at increased concentration and 3 were at lower concentration in the tears of test lens wearers compared to the control lens group. Bootstrap clustering confirmed these findings, showing 3 similar clusters to the original sample groups which separated people wearing control lenses from those wearing TestLens1 or TestLens2 with 83% accuracy and between TestLens1 and TestLens2 with 45% accuracy. Permutation testing identified 5 proteins that had significantly changed in concentration between people wearing TestLens2 and Control lenses. There was no difference in protein concentrations between those subjects who experienced a CIE and those who did not. Conclusion Wearing contact lenses of different ionicities can affect the concentration of proteins in the tear film. The current study did not find any associations of the concentration of proteins with CIEs. Future tests with increased sample size are needed to establish any relations between these changes and clinical performance.

The Human Ocular Surface Microbiome and Its Associations with the Tear Proteome in Dry Eye Disease.

Although dry eye disease (DED) is one of the most common ocular surface diseases worldwide, its pathogenesis is incompletely understood, and treatment options are limited. There is growing evidence that complex interactions between the ocular surface microbiome (OSM) and tear fluid constituents, potentially leading to inflammatory processes, are associated with ocular surface diseases such as DED. In this study, we aimed to find unique compositional and functional features of the OSM associated with human and microbial tear proteins in patients with DED. Applying whole-metagenome shotgun sequencing of forty lid and conjunctival swabs, we identified 229 taxa, with Actinobacteria and Proteobacteria being the most abundant phyla and Propionibacterium acnes the dominating species in the cohort. When DED patients were compared to controls, the species Corynebacterium tuberculostearicum was more abundant in conjunctival samples, whereas the family Propionibacteriaceae was more abundant in lid samples. Functional analysis showed that genes of L-lysine biosynthesis, tetrapyrrole biosynthesis, 5-aminoimidazole ribonucleotide biosynthesis, and the super pathway of L-threonine biosynthesis were enriched in conjunctival samples of controls. The relative abundances of Acinetobacter johnsonii correlated with seven human tear proteins, including mucin-16. The three most abundant microbial tear proteins were the chaperone protein DnaK, the arsenical resistance protein ArsH, and helicase. Compositional and functional features of the OSM and the tear proteome are altered in patients with DED. Ultimately, this may help to design novel interventional therapeutics to target DED.

Experimental design considerations for studies of human tear proteins

PurposeHuman tears contain abundant, diverse sets of proteins that may serve as biomarkers of ocular surface health. There is a need for reproducible methods that consider multiple factors influencing the tear proteome, in addition to the variable of interest. Here we examined a workflow for proteomic analysis of tear proteins without the need to pool tear samples from multiple individuals, thus allowing for analyses based on individual factors, and increasing opportunities for protein biomarker discovery. MethodsTears were collected by Schirmer strip following topical ocular anesthetic application then individually stored at −80 °C prior to processing for proteomics. Tear proteins were extracted from Schirmer strips, digested using suspension trapping spin columns (S-Trap), and labeled with high multiplicity tandem mass tags (TMT). Peptide digests were then extensively fractionated by two-dimensional chromatography and analyzed by mass spectrometry to identify and measure changes in protein abundance in each sample. Analysis of select samples was performed to test protocols and to compare the impact of clinically relevant parameters. To facilitate comparison of separate TMT experiments, common pool samples were included in each TMT instrument run and internal reference scaling (IRS) was performed. ResultsDifferences in subsets of tear proteins were noted for: geographic site of tear collection, contact lens use, and differences in tear fluid volume among individuals. ConclusionThese findings demonstrate that proteomic analysis of human tear proteins can be performed without the need to pool samples, and that development of analytic workflows must consider factors that may affect outcomes in studies focused on diverse clinical samples.

Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium.

Lacripep is a therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with syndecan-1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1-mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 μM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 μM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.

Investigation of the global protein content from healthy human tears

Considering absence of invasiveness and side effects, tears emerge as a particularly attractive fluid for biomarker discovery and therefore for daily clinical use. However, to date this fluid remains poorly studied in healthy condition. Here, we present an updated in-depth characterisation of the human healthy tear protein composition using proteomics approach. Both eyes of 8 healthy controls (4 men and 4 women, average age: 38 ± 18) were collected using the Schirmer's strip method. After liquid digestion and off-gel electrophoresis fractionation, three independent proteomics analyses were performed on a LTQ-Orbitrap Velos Pro. Globally, 1351 proteins were identified with 2 unique peptides and 1% FDR. Gene ontology analyses showed up that 39% of the tear proteins were enzymes, with high numbers of dehydrogenases, phosphatases, kinases and ligases. Immunoglobulins, serpins and 14-3-3 domains proteins also emerged as the main tear protein families. The glycolysis and the coagulation and complement cascades, which were already shown in tears as involved in ocular and systemic diseases, were highlighted performing pathway analyses. Our study therefore complements the existing data on healthy tears proteome. Nevertheless, extensive studies for deeply and definitively characterise this promising fluid are required in the near future in order to be able to routinely use this fluid in clinics. A better understanding of its protein content will probably open new avenues in the biomarker discovery and clinical practice in the near future.

Improvement of Surface Enhanced Raman Spectroscopy By Analyte Molecules Covering with Graphene

Introduction Laser exitation at surface enhanced Raman spectroscopy (SERS) has been found to damage analyte molecules causing changes in their spectra and making it difficult to perform clear qualitative and quantitative analysis [1]. In the present work, we have prevented the destruction of different organic molecules during SERS analysis by their covering with graphene (Gr). Experimental The fabrication of solid substrates demonstrating SERS-activity included two steps of liquid processing: (i) formation of porous silicon (PS) template by anodization of n+-Si wafer in HF-based solution; (ii) electroless deposition of Ag on PS from the aqueous solution of AgNO3 and C2H5OH [2]. Ag was chosen due to its strongest plasmonic properties while the PS template provided nanostructuring Ag deposit. Therefore, combination of these materials led to the enormous enhancement of Raman signal during scattering [2, 3]. Several organic analytes were used to be studied by SERS: dye R6G, porphyrine CuTMpyP4 and human tear proteins (lactoferrine and lizocime). The PS/Ag substrates were immersed into 10-6 – 10-12M solutions of the analytes for 2 h to provide an adsorption of the organic molecules. Gr was grown on Cu foil by the thermal decomposition of methane and acetylene and then transferred onto the PS/Ag and PS/Ag/analyte substrates. Raman spectra were registered on the 3D scanning confocal Raman microscope Confoteq NR500 equipped with 473 nm laser (SOL Instruments, Belarus). Morphology study of the experimental samples was performed with a scanning electron microscope (SEM) Hitachi-4800 (Chiyoda-ku, Japan) provided 1 nm resolution. Results and Discussion Mapping at the Raman scattering of PS/Ag/Gr showed that transferred Gr presents an alternation of single- and multilayered sheets. SEM analysis revealed dimensions and spatial distribution of the Ag nanostructures in the SERS-active substrates are not affected by the covering with Gr (fig. 1). However, there are some Gr wrinkles, which can influence on SERS-activity. Figure 2 shows SERS-spectra of R6G molecules adsorbed on the PS/Ag substrate and covered with the single and multilayered Gr. It is well seen, the multilayered Gr hinders bands of the analyte in contrast to the single Gr. The areas of the PS/Ag/R6G/single Gr were studied by Raman scattering in comparison with the PS/Ag/R6G (fig. 3). Spectrum of the samples free of Gr demonstrate disappearing/fluctuating some R6G characteristic peaks due to photoinduced desorption or chemical reactions with the analyte molecules. Moreover, there are observed broad bands related to the reaction of Ag and products of analyte combustion under laser excitation [4]. On the other hand, Gr-covered samples demonstrate clear peaks typical for R6G molecules. Since Gr presents material, which is inert and resistant to oxygen from environment, it prevents destruction, physical and chemical changes of the underlying organic molecules. The similar results were obtained for CuTMpyP4, lactoferrine and lizocime at all concentrations used in the present work. Conclusion Single Gr cover of organic molecules adsorbed on the SERS-active substrates prevents their destruction or physic-chemical changes. It provides clear determination and concentration estimation of the analyte in aqueous solution. The results are very promising for the analysis of liquids of human body containing molecules, which have similar SERS-spectra.

Quantitation of 47 human tear proteins using high resolution multiple reaction monitoring (HR-MRM) based-mass spectrometry

Tear proteins are intimately related to the pathophysiology of the ocular surface. Many recent studies have demonstrated that the tear is an accessible fluid for studying eye diseases and biomarker discovery. This study describes a high resolution multiple reaction monitoring (HR-MRM) approach for developing assays for quantification of biologically important tear proteins. Human tear samples were collected from 1000 subjects with no eye complaints (411 male, 589 female, average age: 55.5±14.5years) after obtaining informed consent. Tear samples were collected using Schirmer's strips and pooled into a single global control sample. Quantification of proteins was carried out by selecting "signature" peptides derived by trypsin digestion. A 1-h nanoLC-MS/MS run was used to quantify the tear proteins in HR-MRM mode. Good reproducibility of signal intensity (using peak areas) was demonstrated for all 47 HR-MRM assays with an average coefficient of variation (CV%) of 4.82% (range: 1.52-10.30%). All assays showed consistent retention time with a CV of less than 0.80% (average: 0.57%). HR-MRM absolute quantitation of eight tear proteins was demonstrated using stable isotope-labeled peptides. In this study, we demonstrated for the first time the technique to quantify 47 human tear proteins in HR-MRM mode using approximately 1μl of human tear sample. These multiplexed HR-MRM-based assays show great promise of further development for biomarker validation in human tear samples. Both discovery-based and targeted quantitative proteomics can be achieved in a single quadrupole time-of-flight mass spectrometer platform (TripleTOF 5600 system).

Lacritin-mediated regeneration of the corneal epithelia by protein polymer nanoparticles.

The avascular corneal epithelium plays an important role in maintaining normal vision and protecting the corneal interior from environmental infections. Delayed recovery of ocular wounds caused by trauma or refractive surgery strengthens the need to accelerate corneal wound healing and better restore the ocular surface. To address this need, we fused elastin-like polypeptide (ELP) based nanoparticles SI with a model mitogenic protein called lacritin. Lacritin fused at the N-terminus of the SI diblock copolymer is called LSI. This LSI fusion protein undergoes thermo-responsive assembly of nanoparticles at physiologically relevant temperatures. In comparison to ELP nanoparticles without lacritin, LSI showed potent signs of lacritin specific effects on a human corneal epithelial cell line (HCE-T), which included enhancement of cellular uptake, calcium-mediated signaling, and closure of a scratch. In vivo, the corneas of non-obese diabetic mice (NOD) were found to be highly responsive to LSI. Fluorescein imaging and corneal histology suggested that topical administration of LSI onto the ocular surface significantly promoted corneal wound healing and epithelial integrity compared to mice treated with or without plain ELP. Most interestingly, it appears that ELP-mediated assembly of LSI is essential to produce this potent activity. This was confirmed by comparison to a control lacritin ELP fusion called LS96, which does not undergo thermally-mediated assembly at relevant temperatures. In summary, fusion of a mitogenic protein to ELP nanoparticles appears to be a promising new strategy to bioengineer more potent biopharmaceuticals with potential applications in corneal wound healing.

Effect of contact lens solutions on the antimicrobial efficacy of human tear proteins extracted from contact lenses

Purpose: This group previously reported that proteins recovered from conventional hydrogel contact lenses retain their antimicrobial function, with the extent of this activity depending on the care solution used to treat lenses during wear. This study used tear proteins recovered from PureVision silicone hydrogel lenses to examine how antimicrobial activity is affected by carrying out the assays directly in a disinfecting solution containing Polyaminopropyl Biguanide and Polyquaturnium-1, as well as the individual components of the multipurpose solution (MPS).

In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.

Preservation of Human Tear Protein Structure and Function by a Novel Contact Lens Multipurpose Solution Containing Protein-Stabilizing Agents

Tear film proteins have antimicrobial and other functions that may be lost after denaturation during contact lens wear. A new multipurpose solution has recently become available (Biotrue, Bausch + Lomb Inc., Rochester, NY), which contains protein-stabilizing agents including hyaluronic acid, poloxamine, and sulfobetaine 10, the latter used previously as a laboratory tool to renature proteins. We examine whether this new multipurpose solution formulation can prevent the denaturation of human lactoferrin and lysozyme at physiologic levels in response to a powerful denaturing challenge. Human lactoferrin and lysozyme were treated with sodium dodecyl sulfate (SDS) either with or without an investigational version of the new multipurpose solution (without its two disinfectant agents) (investigational multipurpose solution [iMPS]). The structure was assessed by native-polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorometry; additionally, antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus was measured. The iMPS prevented an SDS-induced shift in the native-PAGE banding position of lactoferrin. The SDS treatment substantially altered the lactoferrin DSC and fluorescence spectra, indicating that the protein had denatured. This change did not occur in the presence of iMPS. Lactoferrin and lysozyme showed antibacterial and bacteriolytic activity, which was abolished after SDS treatment; this loss of activity did not occur for proteins treated with iMPS. These data clearly show that the iMPS prevents the denaturation of physiologic levels of human lactoferrin and lysozyme by the strongly denaturing surfactant SDS and that stabilized proteins retain their function. We conclude that this solution has the capacity to stabilize the structure and function of tear proteins.

Human Tear Protein Analysis Enabled by an Alkaline Microfluidic Homogeneous Immunoassay

The ability to probe the protein content of human tear fluid has enormous potential for deepening our understanding of ocular and systemic disease pathology and enabling novel noninvasive tear-based diagnostic technologies. To overcome current challenges in tear proteomic measurements, we report on the first microfluidic homogeneous immunoassay capable of making rapid, quantitative, and specific measurements of endogenous tear protein biomarkers in human tear fluid. Lactoferrin (Lf) is a tear-specific biomarker for Sjögren's syndrome (SS), a serious systemic autoimmune disease currently diagnosed through rudimentary volumetric and surface chemistry measurements and an invasive lip biopsy. We detail optimization of a homogeneous electrophoretic immunoassay for Lf in <1 μL of tear fluid at clinically relevant concentrations. In particular, we present assay development details and a final assay that enables quantification of Lf in <5 s in a clinically relevant range for SS diagnostics. Characterization suggests the on-chip assay is accurate to within 15% of ELISA, specific (<15% nonspecific signal), and with a lower limit of detection of 3 ± 2 nM Lf in human tear matrix. Additionally, we develop and characterize a protocol for eluting proteins from nitrocellulose Schirmer strips, the clinical de facto standard for tear collection and storage. We relate on-chip measured Lf concentrations back to ocular surface concentrations for the first time to our knowledge. Taken in sum, this work details important steps toward (1) expanding the set of proteins quantified by electrophoretic immunoassays to encompass a wider range of isoelectric points than has been reported, (2) creating a first-in-kind translatable assay with clinical relevance to SS diagnostics, and (3) expanding the analytical toolkit available for rapid tear protein measurements, as is relevant to the advancement of basic research and clinical medicine.

The importance of fine dust for the development of an environmentally induced dry eye

Abstract Purpose Environmental conditions promote dry eye by destruction of tear fluid and induction of cell damage. Fine dust is not only present in an urban polluted environment, but also in a cigarette fume or candle light dust loaded home environment. We wanted to determine, if fine dust affects the tear fluid components and conjunctival cells. Methods Human tear fluid from volunteers was obtained and incubated with fine dust suspensions. Possible tear protein changes were analysed by polyacrylamidegel‐electrophoresis (PAGE). Cultivated human conjunctival cells (CHANG‐cells) were incubated with fine dust suspensions as well as exposed to ultraviolet light (UV‐A). Cytomorphological changes and cell vitality were analysed via CASY1 Cell Counter and MTS‐assay. Results Fine dust suspensions destroy tear fluid proteins, particularly in combination with UV‐A‐light irradiation. Moreover, the incubation of human conjunctival cells with fine dust suspensions leads to a statistically significant decrease of cell vitality, depending on the fine dust concentration and incubation period. Additional UV‐A‐irradiation induces a further reduction of cell vitality. Conclusion Due to the alterations of human tear proteins and conjunctival cells caused by fine dust and exaggerated by UV‐A‐irradiation, fine dust can also be responsible for the development of an environmentally induced dry eye.

Applying Magnetic Bead Separation / MALDI-TOF Mass Spectrometry to Human Tear Fluid Proteome Analysis

The proteins and peptides in tears play an important role in preserving the integrity and stability of the ocular surface. Proteomic analysis of tear films will enable us to detect early biological markers of eye diseases, however, it is often hampered by the small amount of tear volume and the low protein concentration. Here we adopted magnetic bead-based purification (ClinProt system) followed by matrix-assisted laser desorption/ionization timeof- flight mass spectrometry (MALDI-TOF-MS) to profile human tear proteins. Basal and reflex tear fluids were collected from normal healthy volunteers using glass microcapillary tubes. Reversed phase (C8) and weak cation exchange (WCX) magnetic beads were applied to obtain multiple components detected as clear signals. Principal component analysis showed a clear differentiation between basal and reflex tears. Among the key alterations, two markedly increased peaks in the reflex tear fluids at m/z 2422.12 and m/z 2721.29 were subsequently analyzed by tandem MS analysis and their source to be proline-rich protein 4 (PRP4). We conclude that magnetic beadbased separation combined with MALDI-TOF-MS (ClinProt MALDI-TOF) appears to be ideally suited for the first-line screening of peptides and proteins in tears.

A Solid-Phase Assay for the Quantitation of Total Protein Eluted from Balafilcon, Lotrafilcon, and Etafilcon Contact Lenses

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.

Characterisation of Human Tear Proteins Using High-resolution Mass Spectrometry

The proteins found in tears play an important role in maintaining the ocular surface and changes in tear protein components may reflect changes in the health of the ocular surface. Proteomics provides a comprehensive approach for cataloguing all the proteins of the tear proteome, which will help to elucidate disease pathogenesis, make clinical diagnoses and evaluate the influence of medications on the structure, composition and secretion of tear proteins. In this study, an alternative proteomic strategy was investigated to explore the human tear proteome. Tear samples were obtained from patients who had pterygium and were collected on the first day and third day after pterygium surgery. Tears pooled from 6 patients were used in the analysis. Reverse-phase high-pressure liquid chromatograph (RPHPLC) was used as the first step to separate intact proteins into 21 peaks. Each fraction was then tryptic-digested and analysed by nanoLC-nano-ESI-MS/MS to characterise the protein components in each fraction. In total, 60 tear proteins were identified with high confidence, including well-known abundant tear proteins, and tear-specific proteins such as lacritin and proline-rich proteins. Among them, proline-rich protein 5 was found for the first time in tear fluid. A large number of plasma proteins were also observed in tear fluid. The results showed that the proteomic strategy used in this study was successfully applied to analyse tear proteome.

Proteomic analysis of human tears: defensin expression after ocular surface surgery.

Human tear protein profiles were monitored by surface enhanced laser desorption/ionization-time-of-flight mass spectrometry ProteinChip technology (SELDI-TOF ProteinChip) and liquid chromatography-mass spectrometry (LC-MS). Tears were collected from 21 patients scheduled for surgery to remove an ocular surface neoplasm prior to surgery (day 0) and on days 1, 3, and 30 postoperatively. Using this proteomic approach, we verified that three human alpha-defensins (HNP-1, HNP-2, and HNP-3) were significantly up-regulated in their expression after surgery and that their levels decreased to approximately normal by day 30 by which time healing was complete. Further confirmation of the identity of the alpha-defensins in human tears was made by LC purification, trypsin digestion, and ESI-MS/MS analysis of their tryptic digests. The concentrations of HNP-1 and HNP-2 were determined and shown to be markedly increased after ocular surface surgery. The results of the study suggest that human alpha-defensins HNP-1, HNP-2, and HNP-3 are up-regulated after surgery, and may in addition to their antimicrobial properties have an important role in wound healing.

Variability of tear protein levels in normal young adults: diurnal (daytime) variation.

To investigate the diurnal variation (daytime changes) of human tear protein levels, and to compare the results obtained by two sampling methods, the yawn and eye-flush methods.

Variability of tear protein levels in normal young adults: between-day variation.

An adequate knowledge of physiological variation is important for valid comparative studies of tear proteins. The aim of this study was to investigate the between-day variation of the human tear protein levels, including the total protein concentration (TPC) and the levels of major protein fractions. Two sampling methods, the yawn and the eye-flush methods, were used and compared. TPC was determined by the Bradford method. The major protein fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and their levels were determined by scanning densitometry after SDS-PAGE. The tear protein levels were monitored for 3 days. The between-day differences in the levels of TPC and the individual protein fractions were not statistically significant in either sampling method, but the variations of some proteins were large and would be clinically significant. Different variations were observed in different proteins. The variations in serum albumin were large, up to 61% and 113% in the yawn and eye-flush methods respectively. The variations in lactoferrin, tear-specific prealbumin and lysozyme were relatively small in the yawn method. The variations in protein levels obtained by the eye-flush method were generally higher than by the yawn method. Although the between-day differences in tear protein levels were not statistically significant, the variations in some proteins would be large in magnitude. The variability of tear protein levels obtained by the eye-flush method was larger than that by the yawn method. Therefore, caution should be taken if the eye-flush method is used for sampling tears for quantitative analysis of tear proteins, although it is easier to collect tear samples using this method. The results will be useful to exclude normal variation in tear protein levels when comparing pre- and post-therapeutic tear protein levels in eyes treated for tear-related abnormalities.

Analysis of Human Tear Fluid Components, Inhibiting Protein Adhesion to Plastic Surfaces

In a previous paper we reported the presence of components in human tear fluid that block the interaction of proteins with plastic surfaces, interfering with tear protein ELISA and proposed the term coating inhibiting activity. The purpose of the study presented here was to further analyse these components. Coating inhibitory activity in human reflex tears was analysed by lectin affinity chromatography, using the agarose bound lectin Artocarpus integrifolia agglutinin (Jacalin), gel filtration, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), blotting and Jacalin staining. For coating inhibitory activity assay in experimental tear samples, the binding of the protein Avidin-conjugated horseradish peroxidase to the polystyrene surface of ELISA micro-titer plate wells, preincubated with the experimental tear samples was measured. In addition, tears were incubated with scrapings of the ELISA plates used in the assay and with six different types of contact lenses (two rigid gas permeable and four hydrogel soft contact lenses) for analysis of adsorbed components. Lectin affinity chromatography of tears yielded a Jacalin-binding and a non-Jacalin-binding preparation, both exhibiting coating inhibitory activity but representing chemically different preparations as observed by SDS–PAGE. After performing gel filtration, coating inhibitory activity eluted with similar retention in both preparations. In fractions exhibiting activity, tear proteins of low molecular weight (< 40 kDa) were detected. Among these, two Jacalin-binding glycoproteins were detected; a major component of approximately 28 kDa and a somewhat smaller minor component. All low molecular weight components were also detected on the scrapings, incubated with tears. The possibility that coating inhibitory activity in tears might reside in a component of larger molecular size can however not be excluded. The human tear proteins secretory Immunoglobulin A, lactoferrin and lysozyme are not involved in coating inhibition. On one of the two rigid gas permeable contact lenses incubated with the tears, the 28 kDa glycoprotein was detected. From the data obtained in our study we conclude that coating inhibitory activity in tears seems to be associated with multiple components of low molecular weight.