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 μL). In a study involving samples from 33 healthy subjects, aliquots (3 μL) 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.

[1]  Y. Konttinen,et al.  Tear fluid plasmin activity of dry eye patients with Sjögren's syndrome. , 2009, Acta ophthalmologica Scandinavica.

[2]  R. López-Solís,et al.  Use of Polyurethane Minisponges to Collect Human Tear Fluid , 2006, Cornea.

[3]  Maciej Kaczmarski,et al.  IL-8 and IFN-γ in Tear Fluid of Patients with Cystic Fibrosis , 2006 .

[4]  J. P. Gilbard The diagnosis and management of dry eyes. , 2005, Otolaryngologic clinics of North America.

[5]  M. Kinoshita,et al.  Decreased mucin concentrations in tear fluids of contact lens wearers. , 2005, Journal of pharmaceutical and biomedical analysis.

[6]  R. Sack,et al.  Characterization of the in vivo forms of lacrimal‐specific proline‐rich proteins in human tear fluid , 2004, Proteomics.

[7]  K. Tsubota,et al.  Abnormal protein profiles in tears with dry eye syndrome. , 2003, American journal of ophthalmology.

[8]  M. Dana,et al.  Proteomic analysis of human meibomian gland secretions , 2003, British Journal of Ophthalmology.

[9]  Hitoshi Watanabe Significance of Mucin on the Ocular Surface , 2002, Cornea.

[10]  B. Starcher,et al.  A ninhydrin-based assay to quantitate the total protein content of tissue samples. , 2001, Analytical biochemistry.

[11]  A J Bron,et al.  Diagnosis of dry eye. , 2001, Survey of ophthalmology.

[12]  C. Baudouin The pathology of dry eye. , 2001, Survey of ophthalmology.

[13]  P. Cho,et al.  Variability of tear protein levels in normal young adults: between-day variation , 2000, Graefe's Archive for Clinical and Experimental Ophthalmology.

[14]  A. Solomon,et al.  The Diagnosis and Management of Dry Eye: A Twenty-five–Year Review , 2000, Cornea.

[15]  S. Pflugfelder,et al.  Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjögren's syndrome keratoconjunctivitis sicca. , 1999, Current eye research.

[16]  M. Komatsu,et al.  Decreased tear lactoferrin concentration in patients with chronic hepatitis C , 1999, The British journal of ophthalmology.

[17]  A. Subramanian,et al.  A red-dot-blot protein assay technique in the low nanogram range. , 1999, Analytical biochemistry.

[18]  R. Beuerman,et al.  The pathology of dry eye: the interaction between the ocular surface and lacrimal glands. , 1998, Cornea.

[19]  B. Holden,et al.  Fibronectin in the tear film. , 1996, Investigative ophthalmology & visual science.

[20]  T. Hamano,et al.  Ocular surface damage and tear lactoferrin in dry eye syndrome , 1994, Acta ophthalmologica.

[21]  A. Kijlstra,et al.  Identification of lectin binding proteins in human tears. , 1991, Investigative ophthalmology & visual science.

[22]  A. Kijlstra,et al.  SDS-Minigel electrophoresis of human tears. Effect of sample treatment on protein patterns. , 1991, Investigative ophthalmology & visual science.

[23]  D. Wilson,et al.  Changes in the polypeptide composition of mouse parotid glands after stimulation of secretion and DNA synthesis by isoproterenol. , 1986, Biochimica et biophysica acta.

[24]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[25]  A. Berta Collection of tear samples with or without stimulation. , 1983, American journal of ophthalmology.

[26]  J. Baum,et al.  Basic tear flow. Does it exist? , 1980, Ophthalmology.

[27]  R. López-Solís,et al.  Effect of sodium hydroxide upon protein binding to filter paper disks and its significance in the determination of protein in colorimetric and radioisotopic assays. , 1979, Analytical biochemistry.

[28]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[29]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[30]  S. Klyce,et al.  Determination of tear volume and tear flow. , 1966, Investigative ophthalmology.

[31]  Oliver H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[32]  F. Paulsen Cell and molecular biology of human lacrimal gland and nasolacrimal duct mucins. , 2006, International review of cytology.

[33]  B. Żelazowska,et al.  IL-8 and IFN-gamma in tear fluid of patients with cystic fibrosis. , 2006, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[34]  P. Sibony,et al.  3 6 0-2 6 0-3 5 0-360-260-1507 8-5 9-IgA IgG , 2005 .

[35]  Ronald E. Smith The tear film complex: pathogenesis and emerging therapies for dry eyes. , 2005, Cornea.

[36]  J. Whitcher,et al.  Tear lysozyme activity in frozen Schirmer strips and salivary gland biopsy as parameters of lacrimal gland function. , 1999, Ocular immunology and inflammation.

[37]  R. Farris The diagnosis and management of the dry eye. , 1997, Journal of ophthalmic nursing & technology.

[38]  G. L. Peterson Determination of total protein. , 1983, Methods in enzymology.

[39]  S. Bramhall,et al.  A simple colorimetric method for determination of protein. , 1969, Analytical biochemistry.