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Immunoassay of Human Tear Lysozyme
IMMUNOASSAY OF HUMAN TEAR LYSOZYME DHAN KRISHNA SEN, M.S., AND GAUTAM SARUP SARIN, B.Sc.
New Delhi, India
Lysozyme, discovered by Fleming;' is widely distributed in nature. Mammalian sources include tears, saliva, serum, neutrophils, macrophages, nasal secretions, and urine.! Alteration in the lysozyme level in human tears has been observed in some ocular diseases, 3-10 so its measurement can be of considerable diagnostic value. Our purpose was twofold: (1) to determine whether the immunodiffusion technique" could be used to estimate tear lysozyme levels; and (2) to investigate whether there is any alteration in tear lysozyme level in patients with trachoma. SUBJECTS AND METHODS
We chose 114 healthy subjects (mean age, 32.6 years) with no ocular or systemic disease seen in the outpatient department between September 1979 and November 1979. They came here mainly for refraction. There were 56 males (mean age, 36.8 years) and 58 females (mean age, 28.6 years). The age distribution of the subjects is given in Table 1. We also selected 30 patients with trachoma (mean age, 34.0 years) treated in the outpatient department during the same period. There were 13 males (mean age, 34.5 years) and 17 females (mean age, 33.7 years). The diagnosis of trachoma was made by slit-lamp examination. The clinical criteria used for the diagnosis were the presence of mature follicles in the upper tarsal conjunctiva, progressive
From the Department of Ophthalmology, Maulana Azad Medical College, and Guru Nanak Eye Centre, New Delhi, India. Reprint requests to D. K. Sen, M.S., V/4, Maulana Azad Medical College Campus, Kotla Road, New Delhi-llOOO2, India.
pannus in the upper part of the cornea, and early signs of cicatrization of the follicles visible with the slit lamp. We collected tear samples by a method described previously," and stored them at -20°C until assayed. We quantified lysozyme by a single radial immunodiffusion technique originally described by Mancini, Carbonera, and Heremans.!' We obtained monospecific rabbit antihuman-lysozyme-serum and the reference standard (Kontrollogen E) from the Behring Institute, West Germany. The manufacturer expresses the lysozyme content of Kontrollogen E as micrograms per milliliter by reference to standard curves prepared with crystalline eggwhite lysozyme (EL). Immunodiffusion plates-We melted 1 % (wt/vol) Special Agar-Noble, buffered in 0.045 M sodium phosphate, pH 7.4 (containing 0.1 M sodium chloride and 0.1 %, wt/vol, sodium azide) in a boiling water bath and then transferred it to a 56°C water bath. We added 0.1 ml of the antiserum to 3 ml of melted agar at 56°C and mixed thoroughly. We poured the agar-antiserum mixture evenly onto a clean glass plate (75 mm x 25 mrn) and placed it on a horizontal surface, using a TABLE 1 DISTRIBUTION OF TEAR LYSOZYME IN NORMAL SUBJECTS
Age (yrs)
No. of Subjects
s15
20 37 28 21 8
16-30 31-45 46-60 2:61
AMERICAN JOURNAL OF OPHTHALMOLOGY 90:715-718, 1980
Mean ± S.D. (mg/ml) 0.8 1.6 1.5 1.1 1.0
± 0.3
± 0.7 ± 0.6 ± 0.5 ± 0.4
Coefficient of Variation (%) 37.50 43.75 40.00 45.45 40.00 715
716
AMERICAN JOURNAL OF OPHTHALMOLOGY
leveling table and a spirit level. We removed air bubbles with a Pasteur pipette. We allowed the agar gel to set at room temperature (24°C). We did the lysozyme assay the same day we prepared the plates. Selection of antiserum concentration -We ascertained the most appropriate antiserum concentration by testing a series of antiserum concentrations in agar gel, choosing the least amount of specific antiserum that gave easily readable ring precipitates. We found this to be 0.1 ml for 3 ml of melted agar. Dilution of tear samples-As the lysozyme concentration in tears is high, we needed to dilute the tear samples to get a clearly defined ring precipitate. Using a qualitative gel diffusion technique;" we found a dilution of 1:25 was satisfactory for the purpose. Lysozyme assay-We cut small antigen wells with absolutely vertical sides with a gel punch, making sure that no cracks developed at the edges. The wells were 2.4 mm in diameter and 12 mm apart. We removed the agar plugs with a suction device. We measured a 4-I..d tear sample with a Hamilton syringe and diluted it with 96 fJ.I of 0.045 M sodium phosphate buffer (pH 7.4) in a 10 mm x 75 mm test tube. We placed 5 fJ.I of diluted tear sample in the antigen wells after mixing. We placed three dilutions of referencestandard known lysozyme concentrations on each plate.
NOVEMBER, 1980
We placed the loaded agar plates in a moist chamber at room temperature (24°C) until diffusion was complete, that is, when daily measurements showed that the precipitation ring failed to increase in size. We measured the diameter of each ring to the nearest 0.1 mm with a magnifying glass with a micrometer scale. We viewed the agar plate against a black background and illuminated it obliquely. We held the plate with the glass side toward the viewer and placed the scale firmly against the glass surface. We used the average of the two perpendicular readings as the measurement for each diffusion ring. When we constructed a standard curve by plotting the squares of the ring diameters against the concentration of the standard solutions, using .l-cm graph paper, we found that it was linear. We constructed a standard curve for each plate and determined the concentration of lysozyme in tear samples with reference to the standard curve. We expressed values as milligrams of lysozyme per milliliter of tears. RESULTS It was essential to test the reproducibility of the method. We therefore repeated the assay of lysozyme four times in each of the tear samples from ten different subjects. Results of an analysis of variance are shown in Table 2. The mean sum of squares caused by variation between replicates was less than the mean sum of
TABLE 2 ANALYSIS OF VAffiANCE
Source of Variation
df
Total Sum of Squares
Mean Sum of Squares
Between subjects Between replicates Error Total
9 3 27 39
9.080 0.017 0.243 9.340
1.009 0.006 0.009
VOL. 90, NO. 5
IMMUNOASSAY OF HUMAN TEAR LYSOZYME
squares due to error. We concluded, therefore, that there was no significant difference between the replicates and that the test results were reproducible. In normal subjects, the value of lysozyme was 1. 3 ± 0.6 mg/ml. There was no significant difference (P > .2) between the level in males (1.4 ± 0.7 mg/ml) and that in females (1.3 ± 0.7 mg/ml). The lysozyme levels in different age groups are shown in Table 1. We found no correlation between age and lysozyme level (r = -0.04), although the level in the group aged 16 to 45 years is higher than those of the younger and older age groups. In patients with trachoma, the level of lysozyme was 0.9 ± 0.5 mg/ml (males, 1.1 ± 0.4 mg/ml, females, 0.8 ± 0.5 mg/ml). There was no significant difference in lysozyme level between males and females in this group (P > .2). The level of tear lysozyme was significantly lower (P < .001) in patients with trachoma than in the normal subjects in the comparable age groups. DISCUSSION
Viscometric.!' electrophoretic.v" and turbidimetric" methods have been used to estimate lysozyme level in tears. These techniques were difficult to apply routinely. The bacterial culture used differed in sensitivity to lysozyme. The data obtained by these methods also varied widely and the values were not comparable. 17 Ronen and associates'" described a spectrophotometric procedure, but the most commonly used method for routine determination of lysozyme is still the lysoplate method.I? We found the immunoassay method for the estimation of lysozyme in human tears to be reproducible and sensitive for routine purposes. Bonavida and Sapse" found the lysozyme level in tears to be 1. 7 mg/ml when measured against human tear lysozyme in
717
normal subjects. Ronen and associates" found the level to be 6.1 mg/ml when measured against hen egg lysozyme. However, lysozyme level by the immunoassy method is 1.3 mg/ml when measured against human serum lysozyme, lower than the above values. We know however, that the level of plasma insulin determined by the innumoassay method'! is also low (21 J.l.units/ml) compared to the level obtained by other methods (diaphragm assay method, 40 to 80 uunits/ml, epididymal adipose tissue method, 135 to 680 J.l.units/ml).20,21 Many investigators have found that age has no effect on the tear lysozyme level. 8,10,14,22,23 Some found it to be independent of sex as well. 8,10,22 Mackie and Seal 24 noted a gradual decline of lysozyme level with advancing age. Bonavida and Sapse" found lower levels of lysozyme at the upper and lower extremes of age. Pietsch and Pearlmanf observed that the level of lysozyme fell continuously after the age of 10 years. Mukai'' found that lysozyme reached peak levels in normal human subjects aged 11 to 20 years and tended to decrease thereafter. We found no significant correlation between age and the level of tear lysozyme, although there was a suggestion that tear lysozyme level rises with age and starts falling after the age of 45 years. We also found that tear lysozyme level was unrelated to the sex of the subjects. Tear lysozyme has been found to be absent or at low levels in some ocular diseases.I''? Our study showed that the level is also low in patients with trachoma compared to normal controls. Testing this method by detecting reduction in the lysozyme level in patients with trachoma proved its utility. SUMMARY
We used a single radial immunodiffusion technique to determine the lysozyme content of human tears. The level
718
AMERICAN JOURNAL OF OPHTHALMOLOGY
in healthy subjects with this method was 1.3 mg/ml of tears. In patients with trachoma, the level was low, 0.9 mg/ml of tears. Detecting the reduction in lysozyme level in patients with trachoma proved the utility of this method. REFERENCES 1. Fleming, A.: On a remarkable bacteriolytic element found in tissues and secretions. Proc. R. Soc. Lond. (BioI.) 93:306, 1922. 2. Selinger, D. S., Selinger, R. C., and Reed, W. B.: Resistance to infection of the external eye. The role of tears. Surv. Ophthalmol. 24:33, 1979. 3. McEwen, W. K., and Kimura, S. J.: Filterpaper electrophoresis of tears. I. Lysozyme and its correlation with keratoconjunctivitis sicca. Am. J. Ophthalmol. 39 (Suppl.):200, 1955. 4. Minton, L. R.: Paralimbal ring keratitis and absence of lysozyme in lupus erythematosus. Am. J. Ophthalmol. 60:532, 1965. 5. Sapse, A. T., Bonavida, B., Stone, W., Jr., and Sercarz, E. E.: Human tear lysozyme. III. Preliminary study on lysozyme levels in subjects with smog eye irritation. Am. J. Ophthalmol. 66:76, 1968. 6. van Bijsterveld, O. P.: Diagnostic tests in the sicca syndrome. Arch. Ophthalmol. 82:10, 1969. 7. El-Gammal, M. Y., and Salah, M.: Estimation of tear lysozyme in some eye diseases. Bull. Ophthalmol. Soc. Egypt 64:285, 1971. 8. Hirai, T.: Studies oflysozyme in tears. J. Tokyo Womens Med. Coll. 42:167, 1972. 9. Mukai, M.: Tear protein. Low molecular protein and lysozyme. Acta Soc. Ophthalmol. Jpn. 79:607, 1975. 10. Avisar, R., Menache, R., Shaked, P., Rubinstein, J., Machtey, I., and Savir, H.: Lysozyme content of tears in patients with Sjogren's syndrome and rheumatoid arthritis. Am. J. Ophthalmol. 87:148, 1979. 11. Mancini, G., Carbonera, A. 0., and Heremans, J. F.: Immunochemical quantitation of antigens by single radial immunodiffusion. Int. J. Immunochem. 2:235, 1965. 12. Sen, D. K., Sarin, G. S., Mani, K., and Saha,
NOVEMBER, 1980
K.: Immunoglobulins in tears of normal Indian people. Br. J. Ophthalmol. 60:302, 1976. 13. Ouchterlony, 0.: Antigen-antibody reaction in gels. Acta Pathol. Microbiol, Scand. 26:507, 1949. 14. Regan, E.: The lysozyme content of tears. Am. J. Ophthalmol. 33:600, 1950. 15. Erickson, O. F., Feeney, L., and McEwen, W. K.: Filter-paper electrophoresis of tears. Animal tears and presence of "slow-moving lysozymes." Arch. Ophthalmcl. 55:800, 1956. 16. Miglior, M., Comite, P. D., and Regnetti, E.: Riesame del l'attivita lisozimica delle lacrime umane normale. In Second International Symposium on Fleming's Lysozyme. Milan, Societa Prodotti Antibiotici, 1961. 17. Bonavida, B., and Sapse, A. T.: Human tear lysozyme. II. Quantitative determination with standard Schirmer strips. Am. J. Ophthalmol. 66:70, 1968. 18. Ronen, D. Eylan, E., Romano, A., Stein, R., and Modan, M.: A spectrophotometric method for quantitative determination of lysozyme in human tears. Description and evaluation of the method and screening of60 healthy subjects. Invest. Ophthalmol. 14:479, 1975. 19. Yalow, R. S., and Berson, S. A.: Immunoassay of endogenous plasma insulin in man. J. Clin, Invest. 39:1157, 1960. 20. Vallance-Owen, J., and Hurlock, B.: Estimation of plasma insulin by the rat diaphragm method. Lancet 1:68, 1954. 21. Pfeiffer, E. F., Pfeiffer, M., Ditschuneit, H., and Alin, G.: Clinical and experimental studies of insulin secretions following tolbutamide and metahexamide administration. Ann. N.Y. Acad. Sci. 82:479, 1959. 22. Janke, W., Langmaack, H., and Tiburtius, H.: Bestimmung der lysozymalen Aktivitat der TranenHussigkeit mit klinisch anwendbaser Methode. Klin. Monatsbl. Augenheilkd. 163:366, 1973. 23. James, W. M.: The lysozyme content of tears. Am. J. Ophthalmol. 18:1109, 1935. 24. Mackie, I. A., and Seal, D. V.: Quantitative tear lysozyme assay in units of activity per microlitre. Br. J. Ophthalmol. 60:70, 1976. 25. Pietsch, R. L., and Pearlman, M. E.: Human tear lysozyme variables. Arch. Ophthalmol. 90:94, 1973.
New Delhi, India
Lysozyme, discovered by Fleming;' is widely distributed in nature. Mammalian sources include tears, saliva, serum, neutrophils, macrophages, nasal secretions, and urine.! Alteration in the lysozyme level in human tears has been observed in some ocular diseases, 3-10 so its measurement can be of considerable diagnostic value. Our purpose was twofold: (1) to determine whether the immunodiffusion technique" could be used to estimate tear lysozyme levels; and (2) to investigate whether there is any alteration in tear lysozyme level in patients with trachoma. SUBJECTS AND METHODS
We chose 114 healthy subjects (mean age, 32.6 years) with no ocular or systemic disease seen in the outpatient department between September 1979 and November 1979. They came here mainly for refraction. There were 56 males (mean age, 36.8 years) and 58 females (mean age, 28.6 years). The age distribution of the subjects is given in Table 1. We also selected 30 patients with trachoma (mean age, 34.0 years) treated in the outpatient department during the same period. There were 13 males (mean age, 34.5 years) and 17 females (mean age, 33.7 years). The diagnosis of trachoma was made by slit-lamp examination. The clinical criteria used for the diagnosis were the presence of mature follicles in the upper tarsal conjunctiva, progressive
From the Department of Ophthalmology, Maulana Azad Medical College, and Guru Nanak Eye Centre, New Delhi, India. Reprint requests to D. K. Sen, M.S., V/4, Maulana Azad Medical College Campus, Kotla Road, New Delhi-llOOO2, India.
pannus in the upper part of the cornea, and early signs of cicatrization of the follicles visible with the slit lamp. We collected tear samples by a method described previously," and stored them at -20°C until assayed. We quantified lysozyme by a single radial immunodiffusion technique originally described by Mancini, Carbonera, and Heremans.!' We obtained monospecific rabbit antihuman-lysozyme-serum and the reference standard (Kontrollogen E) from the Behring Institute, West Germany. The manufacturer expresses the lysozyme content of Kontrollogen E as micrograms per milliliter by reference to standard curves prepared with crystalline eggwhite lysozyme (EL). Immunodiffusion plates-We melted 1 % (wt/vol) Special Agar-Noble, buffered in 0.045 M sodium phosphate, pH 7.4 (containing 0.1 M sodium chloride and 0.1 %, wt/vol, sodium azide) in a boiling water bath and then transferred it to a 56°C water bath. We added 0.1 ml of the antiserum to 3 ml of melted agar at 56°C and mixed thoroughly. We poured the agar-antiserum mixture evenly onto a clean glass plate (75 mm x 25 mrn) and placed it on a horizontal surface, using a TABLE 1 DISTRIBUTION OF TEAR LYSOZYME IN NORMAL SUBJECTS
Age (yrs)
No. of Subjects
s15
20 37 28 21 8
16-30 31-45 46-60 2:61
AMERICAN JOURNAL OF OPHTHALMOLOGY 90:715-718, 1980
Mean ± S.D. (mg/ml) 0.8 1.6 1.5 1.1 1.0
± 0.3
± 0.7 ± 0.6 ± 0.5 ± 0.4
Coefficient of Variation (%) 37.50 43.75 40.00 45.45 40.00 715
716
AMERICAN JOURNAL OF OPHTHALMOLOGY
leveling table and a spirit level. We removed air bubbles with a Pasteur pipette. We allowed the agar gel to set at room temperature (24°C). We did the lysozyme assay the same day we prepared the plates. Selection of antiserum concentration -We ascertained the most appropriate antiserum concentration by testing a series of antiserum concentrations in agar gel, choosing the least amount of specific antiserum that gave easily readable ring precipitates. We found this to be 0.1 ml for 3 ml of melted agar. Dilution of tear samples-As the lysozyme concentration in tears is high, we needed to dilute the tear samples to get a clearly defined ring precipitate. Using a qualitative gel diffusion technique;" we found a dilution of 1:25 was satisfactory for the purpose. Lysozyme assay-We cut small antigen wells with absolutely vertical sides with a gel punch, making sure that no cracks developed at the edges. The wells were 2.4 mm in diameter and 12 mm apart. We removed the agar plugs with a suction device. We measured a 4-I..d tear sample with a Hamilton syringe and diluted it with 96 fJ.I of 0.045 M sodium phosphate buffer (pH 7.4) in a 10 mm x 75 mm test tube. We placed 5 fJ.I of diluted tear sample in the antigen wells after mixing. We placed three dilutions of referencestandard known lysozyme concentrations on each plate.
NOVEMBER, 1980
We placed the loaded agar plates in a moist chamber at room temperature (24°C) until diffusion was complete, that is, when daily measurements showed that the precipitation ring failed to increase in size. We measured the diameter of each ring to the nearest 0.1 mm with a magnifying glass with a micrometer scale. We viewed the agar plate against a black background and illuminated it obliquely. We held the plate with the glass side toward the viewer and placed the scale firmly against the glass surface. We used the average of the two perpendicular readings as the measurement for each diffusion ring. When we constructed a standard curve by plotting the squares of the ring diameters against the concentration of the standard solutions, using .l-cm graph paper, we found that it was linear. We constructed a standard curve for each plate and determined the concentration of lysozyme in tear samples with reference to the standard curve. We expressed values as milligrams of lysozyme per milliliter of tears. RESULTS It was essential to test the reproducibility of the method. We therefore repeated the assay of lysozyme four times in each of the tear samples from ten different subjects. Results of an analysis of variance are shown in Table 2. The mean sum of squares caused by variation between replicates was less than the mean sum of
TABLE 2 ANALYSIS OF VAffiANCE
Source of Variation
df
Total Sum of Squares
Mean Sum of Squares
Between subjects Between replicates Error Total
9 3 27 39
9.080 0.017 0.243 9.340
1.009 0.006 0.009
VOL. 90, NO. 5
IMMUNOASSAY OF HUMAN TEAR LYSOZYME
squares due to error. We concluded, therefore, that there was no significant difference between the replicates and that the test results were reproducible. In normal subjects, the value of lysozyme was 1. 3 ± 0.6 mg/ml. There was no significant difference (P > .2) between the level in males (1.4 ± 0.7 mg/ml) and that in females (1.3 ± 0.7 mg/ml). The lysozyme levels in different age groups are shown in Table 1. We found no correlation between age and lysozyme level (r = -0.04), although the level in the group aged 16 to 45 years is higher than those of the younger and older age groups. In patients with trachoma, the level of lysozyme was 0.9 ± 0.5 mg/ml (males, 1.1 ± 0.4 mg/ml, females, 0.8 ± 0.5 mg/ml). There was no significant difference in lysozyme level between males and females in this group (P > .2). The level of tear lysozyme was significantly lower (P < .001) in patients with trachoma than in the normal subjects in the comparable age groups. DISCUSSION
Viscometric.!' electrophoretic.v" and turbidimetric" methods have been used to estimate lysozyme level in tears. These techniques were difficult to apply routinely. The bacterial culture used differed in sensitivity to lysozyme. The data obtained by these methods also varied widely and the values were not comparable. 17 Ronen and associates'" described a spectrophotometric procedure, but the most commonly used method for routine determination of lysozyme is still the lysoplate method.I? We found the immunoassay method for the estimation of lysozyme in human tears to be reproducible and sensitive for routine purposes. Bonavida and Sapse" found the lysozyme level in tears to be 1. 7 mg/ml when measured against human tear lysozyme in
717
normal subjects. Ronen and associates" found the level to be 6.1 mg/ml when measured against hen egg lysozyme. However, lysozyme level by the immunoassy method is 1.3 mg/ml when measured against human serum lysozyme, lower than the above values. We know however, that the level of plasma insulin determined by the innumoassay method'! is also low (21 J.l.units/ml) compared to the level obtained by other methods (diaphragm assay method, 40 to 80 uunits/ml, epididymal adipose tissue method, 135 to 680 J.l.units/ml).20,21 Many investigators have found that age has no effect on the tear lysozyme level. 8,10,14,22,23 Some found it to be independent of sex as well. 8,10,22 Mackie and Seal 24 noted a gradual decline of lysozyme level with advancing age. Bonavida and Sapse" found lower levels of lysozyme at the upper and lower extremes of age. Pietsch and Pearlmanf observed that the level of lysozyme fell continuously after the age of 10 years. Mukai'' found that lysozyme reached peak levels in normal human subjects aged 11 to 20 years and tended to decrease thereafter. We found no significant correlation between age and the level of tear lysozyme, although there was a suggestion that tear lysozyme level rises with age and starts falling after the age of 45 years. We also found that tear lysozyme level was unrelated to the sex of the subjects. Tear lysozyme has been found to be absent or at low levels in some ocular diseases.I''? Our study showed that the level is also low in patients with trachoma compared to normal controls. Testing this method by detecting reduction in the lysozyme level in patients with trachoma proved its utility. SUMMARY
We used a single radial immunodiffusion technique to determine the lysozyme content of human tears. The level
718
AMERICAN JOURNAL OF OPHTHALMOLOGY
in healthy subjects with this method was 1.3 mg/ml of tears. In patients with trachoma, the level was low, 0.9 mg/ml of tears. Detecting the reduction in lysozyme level in patients with trachoma proved the utility of this method. REFERENCES 1. Fleming, A.: On a remarkable bacteriolytic element found in tissues and secretions. Proc. R. Soc. Lond. (BioI.) 93:306, 1922. 2. Selinger, D. S., Selinger, R. C., and Reed, W. B.: Resistance to infection of the external eye. The role of tears. Surv. Ophthalmol. 24:33, 1979. 3. McEwen, W. K., and Kimura, S. J.: Filterpaper electrophoresis of tears. I. Lysozyme and its correlation with keratoconjunctivitis sicca. Am. J. Ophthalmol. 39 (Suppl.):200, 1955. 4. Minton, L. R.: Paralimbal ring keratitis and absence of lysozyme in lupus erythematosus. Am. J. Ophthalmol. 60:532, 1965. 5. Sapse, A. T., Bonavida, B., Stone, W., Jr., and Sercarz, E. E.: Human tear lysozyme. III. Preliminary study on lysozyme levels in subjects with smog eye irritation. Am. J. Ophthalmol. 66:76, 1968. 6. van Bijsterveld, O. P.: Diagnostic tests in the sicca syndrome. Arch. Ophthalmol. 82:10, 1969. 7. El-Gammal, M. Y., and Salah, M.: Estimation of tear lysozyme in some eye diseases. Bull. Ophthalmol. Soc. Egypt 64:285, 1971. 8. Hirai, T.: Studies oflysozyme in tears. J. Tokyo Womens Med. Coll. 42:167, 1972. 9. Mukai, M.: Tear protein. Low molecular protein and lysozyme. Acta Soc. Ophthalmol. Jpn. 79:607, 1975. 10. Avisar, R., Menache, R., Shaked, P., Rubinstein, J., Machtey, I., and Savir, H.: Lysozyme content of tears in patients with Sjogren's syndrome and rheumatoid arthritis. Am. J. Ophthalmol. 87:148, 1979. 11. Mancini, G., Carbonera, A. 0., and Heremans, J. F.: Immunochemical quantitation of antigens by single radial immunodiffusion. Int. J. Immunochem. 2:235, 1965. 12. Sen, D. K., Sarin, G. S., Mani, K., and Saha,
NOVEMBER, 1980
K.: Immunoglobulins in tears of normal Indian people. Br. J. Ophthalmol. 60:302, 1976. 13. Ouchterlony, 0.: Antigen-antibody reaction in gels. Acta Pathol. Microbiol, Scand. 26:507, 1949. 14. Regan, E.: The lysozyme content of tears. Am. J. Ophthalmol. 33:600, 1950. 15. Erickson, O. F., Feeney, L., and McEwen, W. K.: Filter-paper electrophoresis of tears. Animal tears and presence of "slow-moving lysozymes." Arch. Ophthalmcl. 55:800, 1956. 16. Miglior, M., Comite, P. D., and Regnetti, E.: Riesame del l'attivita lisozimica delle lacrime umane normale. In Second International Symposium on Fleming's Lysozyme. Milan, Societa Prodotti Antibiotici, 1961. 17. Bonavida, B., and Sapse, A. T.: Human tear lysozyme. II. Quantitative determination with standard Schirmer strips. Am. J. Ophthalmol. 66:70, 1968. 18. Ronen, D. Eylan, E., Romano, A., Stein, R., and Modan, M.: A spectrophotometric method for quantitative determination of lysozyme in human tears. Description and evaluation of the method and screening of60 healthy subjects. Invest. Ophthalmol. 14:479, 1975. 19. Yalow, R. S., and Berson, S. A.: Immunoassay of endogenous plasma insulin in man. J. Clin, Invest. 39:1157, 1960. 20. Vallance-Owen, J., and Hurlock, B.: Estimation of plasma insulin by the rat diaphragm method. Lancet 1:68, 1954. 21. Pfeiffer, E. F., Pfeiffer, M., Ditschuneit, H., and Alin, G.: Clinical and experimental studies of insulin secretions following tolbutamide and metahexamide administration. Ann. N.Y. Acad. Sci. 82:479, 1959. 22. Janke, W., Langmaack, H., and Tiburtius, H.: Bestimmung der lysozymalen Aktivitat der TranenHussigkeit mit klinisch anwendbaser Methode. Klin. Monatsbl. Augenheilkd. 163:366, 1973. 23. James, W. M.: The lysozyme content of tears. Am. J. Ophthalmol. 18:1109, 1935. 24. Mackie, I. A., and Seal, D. V.: Quantitative tear lysozyme assay in units of activity per microlitre. Br. J. Ophthalmol. 60:70, 1976. 25. Pietsch, R. L., and Pearlman, M. E.: Human tear lysozyme variables. Arch. Ophthalmol. 90:94, 1973.