Download Electrophoresis combined with immunologic identification of

Investigative Ophthalmology & Visual Science, Vol. 30, No. 8, August 1989
Copyright © Association for Research in Vision and Ophthalmology
Electrophoresis Combined with Immunologic
Identification of Human Tear Proteins
P. K. Coyle,* Patrick A. 5ibony,*f and Cynthia Johnson*
The protein content of normal human tears from five subjects was examined by molecular weight
separation using SDS-polyacrylamide gel electrophoresis (PAGE) and by charge separation using
agarose isoelectric focusing (IEF) gels. After separation, specific proteins were identified by immunoblot and immunofixation. Tear proteins examined included albumin, IgA, IgG, prealbumin, lactoferrin,
lysozyme, secretory component and transferrin. These techniques required 1 to 14 /il of unconcentrated tears. We found SDS-PAGE superior to agarose IEF to examine total tear protein pattern, and
silver stain almost ten-fold more sensitive than Coomassie blue stain. Immunologic staining markedly
enhanced protein detection in all tear samples and appeared to offer the definitive method to probe for
a specific protein in tears. In this study prealbumin and a portion of the IgG were present in normal
tears at higher than expected molecular weight, suggesting they were present in complexed form.
Prealbumin and secretory component staining showed marked variability between subjects. These
techniques should be applicable to examine tear proteins in a variety of ocular disease states. Invest
Ophthalmol Vis Sci 30:1872-1878,1989
The role of the tear film in human health and disease has come under increasing scrutiny.1 Accurate
and sensitive characterization of tear components is
crucial to interpreting the changes found in ocular
surface disorders. Yet the techniques applied in tear
studies must be reliable on very small volumes. Although a variety of electrophoretic techniques have
been applied to standardize tear proteins, newer and
more sensitive immunologic probes such as immunoblot and immunofixation have not. In this study
we examined normal human tears for eight protein
components using immunologic probes and electrophoresis based on molecular weight and isoelectric
point separation.
Informed consent was obtained from all individuals
after the nature of the procedures was fully explained.
There were four females and one male aged 23 to 43
years.
Sample Collection
Reflex tears were stimulated with aromatic ammonia and collected using glass capillary tubes gently
inserted into the tear pool of the lower lid margin as
previously reported.2 Care was taken not to touch or
irritate the eye directly. Tears were stimulated until at
least 100 /A had been collected; total collection time
was 2 to 3 min. Final volumes from the five subjects
ranged from 100 to 150 /A. Tears were stored at -20°
or -70°C until use.
Materials and Methods
Subjects
Tear Proteins
Five normal subjects without clinical signs of eye
disease were studied. None were on any medications.
Total protein concentration was measured on 3 nl
of tears using the Bradford method, as previously reported.3 Bovine albumin was used as the protein
standard.
The following proteins were examined in tears
using immunologic identification as outlined below:
albumin, IgA, IgG, lactoferrin, lysozyme, prealbumin, secretory component and transferrin.
From the Departments of *Neurology and fOphthalmology,
Health Sciences Center, SUNY at Stony Brook, Stony Brook, New
York.
Presented in part at the Annual Meeting of the Association for
Research in Vision and Ophthalmology, Sarasota, Florida, May
1-6,1988.
Supported by the National Eye Institute (grant EY-05736).
Submitted for publication: June 6, 1988; accepted February 14,
1989.
Reprint requests: Dr. P. K. Coyle, Department of Neurology,
HSC T-12, SUNY at Stony Brook, Stony Brook, NY 11794.
Tear Electrophoresis
Two different techniques were used to study total
tear proteins. To separate proteins by molecular
weight, fixed protein concentrations of tears were
1872
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No. 8
1873
ELECTROPHORESIS OF HUMAN TEARS / Coyle er ol
electrophoresed on 12% separating SDS-polyacrylamide gels (PAGE) using 5% stacking gels as previously reported.4 Because 12% gels will not give reliable resolution of proteins above 160 kilodaltons
(kD), 7% separating gels were also used to analyze
higher molecular weight tear proteins. Samples were
run both reduced and unreduced, and a Tris-HCl
with glycine buffer system was used. Commercial
molecular weight standards (BRL Inc, Gaithersberg,
MD; and Sigma Chemical Co, St. Louis, MO) were
run in tandem and used to calibrate the molecular
weights of tear bands. Gels were stained with Coomassie blue (CB) or silver as outlined below.
To separate proteins by isoelectric point, fixed volumes of tears were run rather than fixed protein concentrations. In preliminary studies volume standardization produced optimal gel results, whereas protein
standardization gave inconsistent results. Tears were
diluted to 20 /A in deionized water and run on agarose
isoelectric focusing (IEF) gels purchased from IsoLab, Inc. (Akron, OH). For the anode solution 0.5 M
acetic acid was used, and for the cathode solution 0.5
M NaOH. Gels with a pH range of 3-10 were used to
examine total proteins, IgG and prealbumin. Gels
with a pH range of 7-10 were used to examine lysozyme. In all other cases gels from pH 3-7 were used.
Gels were run on the commercial Resolve electrophoresis unit according to the manufacturer's instructions and stained as outlined below. Gels were calibrated by measuring the pH at 1 cm intervals from
the anode using a micro-combination electrode.
Gel Staining
SDS-PAGE gels were stained with CB or silver. For
CB staining 8 ng of total tear protein was run. This
was reduced to 1 ng for silver staining. CB gels were
fixed and stained in 0.1% CB, 50% ethanol, 10% acetic acid for 1 hr and destained in two to three changes
of 30% isopropanol, 10% acetic acid. For silver staining gels were first stained with CB, then washed overnight in four changes of 30% isopropanol, 10% acetic
acid. The staining method of Merril was then used.5
Agarose IEF gels were washed in 5% glycerol, dried at
70°C for 70 min, and then silver-stained using the
Resolve-CSF kit reagents and method (IsoLab, Inc.,
Akron, OH).
Protein Identification
Specific proteins were identified in tears by immunologic probes. For these assays commercial human
protein standards were obtained as follows: albumin
(Sigma Chemical Co.), IgA and IgG (Tago, Inc., Burlingame, CA), lactoferrin, lysozyme and prealbumin
(Calbiochem, La Jolla, CA), secretory component
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(Cappel, Malvern, PA), and transferrin (Jackson Immunoresearch, West Grove, PA). Commercial primary antisera (affinity-purified antibodies were used
when available) were obtained as follows: goat antialbumin, goat anti-IgA and goat anti-IgG (Tago,
Inc.), rabbit anti-lactoferrin (Jackson Immunoresearch), rabbit anti-lysozyme, rabbit anti-prealbumin
and rabbit anti-secretory component (Dako Corp.,
Santa Barbara, CA), and goat anti-transferrin (Cappel). In preliminary experiments cross-reactivity between the standards and antisera was examined by gel
electrophoresis followed by immunoblot. No crossreactivity was noted except for the prealbumin antisera, which reacted with albumin, lactoferrin, lysozyme and transferrin. Prior to use the prealbumin
antisera was preabsorbed with these standards until
all cross-reactivity (by immunoblot) was removed.
Immunoblot was used to identify tear proteins separated by SDS-PAGE.6'7 Five micrograms of total
tear protein were run for all immunoprobes except
for IgG, in which case 9 /*g were used. Proteins were
electrophoretically transferred to a 0.1 ^m nitrocellulose membrane (Schleicher & Schuell, Keene, NH) at
100 vc for 1 hr. The membrane was blocked by exposure to 3% bovine serum albumin-phosphate-buffered saline for 90 min at 37°C and incubated with an
optimal dilution of the primary antisera for 30 min at
37°C and 30 min at room temperature (RT). After
several washes in PBS-Tween, the membrane was
then incubated with horseradish peroxidase-conjugated anti-goat or anti-rabbit IgG antibodies (depending on the species-specific primary antisera) for
1 hr at RT. After washing the membrane was developed using 0.03% diaminobenzidine and 0.015%
H2O2 in deionized water for 10 min at RT, blotted
dry, and photographed on Panatonic-X black and
white film using a Nikon FE 35 mm camera equipped
with a Mikro-Nikkor 55 mm f/2.8 lens and green
filter.
Immunofixation was used to identify tear proteins
separated by agarose-IEF as previously reported.8
Gels were overlayed with a cellulose acetate membrane saturated with undiluted primary antisera for 1
hr at RT. Proteins not bound by antibody were then
washed out of the gel over a 24 to 48 hr period with
0.15 M NaCl, 5% glycerol. After a final wash with 5%
glycerol, the gels were dried and silver-stained as reported above.
Results
Tear Protein Content
Total protein concentration of the five samples fell
within a fairly narrow range: 4.6, 6.2 (two subjects),
6.3 and 6.9 mg/ml.
1874
Vol. 30
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Augusr 1989
CB Stain
Ag Stain
290
230
210-
1175-*
74
68
55'
39
31-
17.5
16.5'
14.5
14
13.5'
4C
i
V*
Fig. 1. (A) Eight micrograms of normal tears were
run on nonreducing 12%
SDS-PAGE and stained
with CB; three representative samples are shown.
Seven bands were routinely
visualized with occasional
additional bands at 31 and
16 kD. (B) One microgram
of normal tear was run as in
A, then stained with silver
(Ag). Three representative
samples are shown. Five additional bands were noted
routinely, with an occasional sample showing a
faint 175 kD band.
0
A
Tear Analysis Using SDS-PAGE
Eight micrograms of total tear proteins were run on
SDS-PAGE and stained with CB (Fig. 1A). Seven
bands were routinely visualized: a very dense 14.5 kD
band, moderately dense bands at 16.5, 17.5 and 68
kD, and faint bands at 55, 74 and 230 to 290 kD.
Additional bands were noted in two samples at 31 kD
and in one sample at 16 kD. Because of the increased
sensitivity of silver staining, tears were run at a total
protein concentration of 1 /ig (Fig. 1B). Silver staining accentuated the seven bands noted with CB as
well as the 31 kD band, which now appeared in four
samples. In addition all samples showed faint but
discrete bands in the region of 13.5, 14, 39, 44 and
210 kD. Two samples showed an additional faint
band at 175 kD. When a 7% separating gel was used
(Fig. 2), there was improved resolution at the top of
the gel with three discrete bands noted from 280-380
kD. However, bands below 35 kD were lost. Reduced
tear samples run on both 7% and 12% separating gels
showed less intense staining of bands at the top of the
gel, with the appearance of a new dense staining at
approximately 80-88 kD (Fig. 2). This would be in
the region where lactoferrin would run.
7%
12%
Protein Identification by Immunoblot
20097.468-
200-
43-
97.4 -
25.7-
68-
I
18.414.3-
43-
N
N
Fig. 2. Nonreduced (N) and reduced (R) tears were electrophoresed on 12% and 7% SDS-PAGE, then silver-stained. Known molecular weight standards are indicated on the left of each gel pair.
Reduced tears show fainter staining at the top of the gel, and a new
band at the approximate molecular weight of lactoferrin (arrow).
The 7% gel gives better resolution of proteins at the top of the gel.
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Tears were electrophoresed on SDS-PAGE, transferred to nitrocellulose and immunostained for specific proteins. The overall patterns noted are outlined
in Table 1. Representative samples are shown in Figure 3 for all proteins separated on 12% gels, and in
Figure 4 for higher molecular weight proteins (IgA,
IgG, secretory component) separated on 7% gels.
There were several unexpected findings. IgG stained
at higher than expected weights (310 and 350 kD) in
addition to the 150 kD for monomeric immunoglobulin (Fig. 4). This was not an artifact of storage, since
fresh tear samples gave identical results. To exclude
the possibility that IgG reagents were reacting with
IgA, specific immunoblot studies were carried out at
several concentrations and documented no cross-reactivity with up to 1 Mg of purified IgA standard.
Prealbumin showed a marked variability in staining
between tear samples. All samples stained faintly at
70 kD, higher than the 1.3 kD reported for prealbu-
1875
ELECTROPHORESIS OF HUMAN TEARS / Coyle er ol
No. 8
Table 1. Tear proteins identified by SDS-PAGE and immunoblot*
Protein molecular
weight (kD)
Tear volume
Protein examined
used (nl)
Protein staining pattern
Albumin
IgA
IgG
Lactoferrin
Lysozyme
Prealbumin
Secretory component
Transferrin
0.8
0.8
1.5
0.8
0.8
0.8
0.8
0.8
one band
one band
three bands
two dense bands
one dense band; two faint bands
two faint bands
two-three bands
one band
Intersubject
variability
minimal
moderate
moderate
minimal
minimal
marked
moderate
minimal
55
260-360
150,310,350
74,81
14.5, 23, 24
31,70
59, 78, 260-360
75
* Tears from five normal subjects were electrophoresed on nonreducng
12% or 7% SDS-PAGE gels, transferred to nitrocellulose, and immunoblotted for eight specific proteins. Molecular weight was calculated by co-elec-
trophoresing known molecular weight standards. Intersubject variability was
judged to be minimal, moderate or marked depending on the degree of
conformity between thefivesamples.
min subunits9 or the 21 to 24 kD reported for tearspecific prealbumin. 10 " Three samples showed an
additional band at 31 kD and in one sample this band
was quite intense. Secretory component in the 7% gel
stained diffusely at 260-360 kD (bound to IgA), as
well as at 78 kD and very faintly at 59 kD (free or
precurser forms).12 Based on comparison of molecular weight and band intensity, it appeared that in the
12% gels the 14.5 kD band in CB- or silver-stained
gels represented lysozyme; the 31 kD band represented prealbumin; the 55 kD band represented albumin; the 68 kD band represented lactoferrin and
possibly prealbumin; the 74 kD band represented
lactoferrin and possibly secretory component; and
the 230 to 280 kD band represented IgA and IgG.
The other CB- and silver-stained bands were not
identified as any of the eight proteins probed for.
Because of the enhanced gel staining noted on reduced tears, an immunoblot for lactoferrin was carried out on the same tear sample nonreduced and
reduced. There was a marked increase in lactoferrin
staining in the reduced sample (data not shown).
(Fig. 5). Multiple bands were noted in all samples
from pH 3.6 to 6.4. Staining was almost homogeneous from 5.0 to 6.0. There were major bands at pH
8.0 and 9.7, and occasional faint bands in the region
ofpH7.3and8.4.
Tear Analysis Using Agarose-IEF
Ten microliters of tears were run on agarose-IEF
gels (pH 3-10) and silver-stained for total protein
Fig. 3. Tears were electrophoresed on nonreducing 12% SDS-PAGE, transferred to nitrocellulose and
immunostained. Specific
proteins examined were albumin (Alb), IgA, IgG, lactoferrin (Lact), lysozyme
(Lys), prealbumin (PAlb),
secretory component (SC)
and transferrin (Trans).
Representative samples for
each protein are shown,
along with the calibrated
molecular weights.
Protein Identification by Immunofixation
Optimal volumes of unconcentrated tears were
electrophoresed on agarose-IEF gels, then immunofixed for specific proteins. The volume for each protein had been determined in preliminary studies as
giving optimal gel results. Tear patterns are noted in
Table 2 and representative samples shown in Figure
6. Single bands were noted for albumin, lysozyme
and transferrin. Lysozyme stained at pH 9.7 in this
gel system, which only measured up to pH 10. This
slightly underestimated its true pH range, which has
been reported as 10 to 11." IgA showed multiple
bands of varying intensity ranging from pH 4.4 to
6.1, whereas IgG by this technique showed a limited
number of very faint bands in the pH range 4.7 to 8.0.
Lactoferrin stained diffusely and very intensely over
the pH 3.9 to 5.3 region. In this less sensitive technique tear prealbumin was highly variable: one subject showed no prealbumin, three had a single band at
270*
200**
240 >•190*-
310*
97-*
79-*
8174"
55
70-
75-**
2 ^
23-*
14.5*
Alb
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IgA
IgG
Lact
Lys
PAlb
SC
Trans
1876
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1989
360260-
pH 4.4 (consistent with the standard) and one subject
had three faint bands at pH 4.4, 4.6 and 4.9. Secretory component showed marked variability when
separated by isoelectric point with obvious variations
in amount of staining. Multiple bands were noted
from pH 4.15 to pH 6.7, with the densest staining in
the pH 5.3 to 5.9 region.
360260-
350-
1507859-
IgA
IgG
SC
Fig. 4. Tears were electrophoresed on nonreducing 7% SDSPAGE, transferred to nitrocellulose and immunostained. Specific
proteins examined on this gel were IgA, IgG and secretory component (SC). Representative samples and calibrated molecular
weights are shown.
Ag Stain
PH
3.6
6.4
7.3
8.0
8.4
9.7
Fig. 5. Ten microliters of normal tears were run on agarose IEF
gels, pH 3-10, and silver-stained. Three representative samples are
shown. The pH gradient measured is noted at the left.
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Vol. 30
Discussion
Tears are made up of an aqueous layer covered by a
thin lipid film. Their chemical composition is quite
complex and includes over sixty proteins and polypeptides as well as electrolytes, enzymes, lipids, metabolites and mucin.13 Tears are produced by the innervated lacrimal gland along with contributions
from minor accessory glands and the conjunctiva.
With a baseline production rate of 1 to 2 jul per minute, and only 5 to 10 ^1 available in the eye at any
given time,14 the absolute volume of tears is quite
small. Therefore reflex tears produced by neural stimulation are generally used to provide a workable volume. Even so, the absolute amount obtainable from
subjects varies, and this can become a limiting factor
in tear studies.
A number of investigators have analyzed tear proteins using a variety of methods. Electrophoretic
studies have included disc electrophoresis,15 electroimmunoassay,16 dissociating and nondissociating
PAGE,11131517"19 agar and agarose gel immunoelectrophoresis,11-15'19'20-23 IEF,1518 and two-dimensional
electrophoresis.11'317 When immunologic means
have been used to identify tear proteins they have not
been used in conjunction with techniques that distinguished molecular weight or isoelectric point. To our
knowledge, the current study is the first systematic
analysis of tear proteins using the newer technology
of silver stain, immunoblot and immunofixation.
These studies were carried out on unconcentrated
tears, avoiding the potential loss of protein. And they
required only 1 to 14 /il volumes, compared to prior
studies which had to pool samples or use up to 100 /ul
oftears.13'15-17-19-21
We found separation of tear proteins by weight
rather than charge to be more useful and interpretable as a screening examination, because many tear
proteins had similar charges. This required two different separating gels (7% and 12%) since proteins
over 160 kD were not sufficiently resolved on the
12% gel. As expected, silver staining was much more
sensitive than CB. Silver staining permitted almost a
10-fold reduction in sample protein, yet resulted in
enhanced detection. However, it was clear that immunologic staining was superior even to silver, and
offered the definitive way to probe for specific tear
proteins. It is simply not reliable to depend on co-
1877
ELECTKOPHORE5IS OF HUMAN TEARS / Coyle er QI
No. 8
Table 2. Tear proteins identified by agarose IEF and immunofixation*
Protein examined
Tear volume
used (pi)
Albumin
one band
multiple bands
faint bands
homogeneous dense stain
one band
one-three faint bands
multiple bands
one band
5
5
14
0.5
3
10
6
10
IgA
IgG
Lactoferrin
Lysozyme
Prealbumin
Secretory component
Transferrin
Protein pH
range
Protein staining pattern
Intersubject
variability
moderate
minimal
minimal
minimal
minimal
marked
marked
moderate
4.5
4.4-6.1
4.7-8.0
3.8-5.3
9.7
4.4, 4.6, 4.9
4.15-6.7
5.1
* Optima) unconccntrated tear volumes from five normal subjects were
electrophorescd on agarose IEF gels and immunofixed for eight specific
proteins. The pH of stained bands was measured using a microelectrode
probe. Intersubject variability between thefivesamples was judged as noted
in Table 1.
electrophoresis to identify proteins, as prior studies
have done. The immunologic staining was able to
enhance detection of all proteins looked at, even
those in low concentration which could not be identified on gels stained for total tear proteins.
Our protein weights and charges were in fairly close
agreement with those reported in the literature although there were some differences. We noted tear
IgA to stain as a diffuse band from 260 to 360 kD on
7% gels. This is close to its expected weight of 380 kD,
since tear IgA is known to be dimeric with an attached joining chain and secretory component.12 It is
likely that some of the IgA entering our SDS-PAGE
gels was partially dissociated. IgG is present at low
levels in tears and is presumed to be monomeric.
However in addition to the 150 kD diffuse band,
there were two consistent bands at 310 and 350 kD.
They did not appear to be due to aggregation, since
the bands ran above an aggregated IgG standard.
Tear IgG has been said to originate from serum.24
Our results suggest that part of the IgG in tears is
routinely complexed. This would support it being
tear-specific and emphasize a possible functional
role. Fullard has pointed out that both lactoferrin and
lysozyme may bind to immunoglobulins.25 We feel
this is unlikely to account for our IgG results since
lactoferrin or lysozyme did not stain in this region. It
is apparent that a large proportion of tear lactoferrin
is complexed, since it was of sufficient weight not to
enter a gel until it was reduced. In the current study
lactoferrin showed a more anodal charge range than
previously reported.1115 We also found differences
from previous reports with regard to tear prealbumin,
which stained at 31 and 70 kD. Although our antiserum was directed against serum prealbumin, in
preabsorption studies we removed all nonspecific
staining. And the isoelectric point values we obtained
for prealbumin were in close agreement with those
reported in the literature. 10 " 15 Our 31 kD band suggests that "protein G" identified by Gachon may actually be prealbumin.17 We could not confirm a
prealbumin 24 kD band. Our 70 kD band may represent joined subunits, since prealbumin, at least in
serum, is known to exist frequently as four joined
subunits.9 Alternatively, it could be that prealbumin
is bound to albumin. This has been reported to occur
for 3 to 7% of prealbumin isolated from human ocular mucus.26 To explain our prominent staining in
tears, however, the percent bound would have to be
much higher.
3.8 *
4.4
...
4.'
4.5-
4.15*
I*
4.9
Fig. 6. Tears were electrophoresed on agarose-IEF
gels, then immunofixed for
the eight specific proteins
noted in Figure 2. Representative samples for each
protein are shown along
with the pH measured for
stained bands.
5.1
5,3 *•
6.0*
67>
—
I
9-7
Alb
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IgA
IgG
Loct
Lys
PAlb
SC
Trans
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1989
1880
incubation, 100 iA normal rabbit serum, 100 y\ antirabbit IgG goat serum and 200 /A 15% polyethylene
glycol were added to each tube, followed by further
incubation at room temperature for 1 hr. After centrifugation at 1600 g for 30 min, radioactivity in the
precipitates was measured by a gamma counter.
Intra-assay as well as inter-assay variations in RIA,
estimated using samples of varied concentrations of
standard hEGF, were within acceptable ranges. The
minimum detectable dose was 0.1 ng/ml. No crossreactivity was observed with rat and mouse EGFs.
Platelet-derived growth factor, human transforming
growth factors-a and -0, human insulin-like growth
factor-I, human insulin, porcine glucagon and ACTH
also showed no cross-reactivities in this assay system.
ng/ml
10-
Vol. 30
OO
5-
Radioreceptorassay (RRA)
OO
Tear
non-reflex reflex
IIIIIIIB
Aqueous
Humor
Fig. 1. Concentrations of hEGF in tears and aqueous humor.
Bars indicate mean values ± standard deviation.
dilution ratio of urine (•—•)
x80 x40 x20 x10
x5
95i
dilution ratio of tears (
x4
x2
x1 xi/2
A431 cells derived from human epidermoid carcinoma were used as the EGF receptor source. Synthetic hEGF was used as standard and substrate for
125
I-hEGF. Standard diluent used for RRA was 50
raM sodium phosphate buffer (pH 7.4) containing 25
mM EDTA, 140 mM NaCl, 0.5% bovine serum albumin and 0.02% sodium azide. A mixture of 200 ^1
test sample or standard hEGF, 100 ix\ 125I-labeled
hEGF and 200 /xl formalin-fixed A431 cell suspension (2 X 105 cells) in each assay tube was incubated
at 25°C for 16 hr. After incubation, 1 ml ice-cold
standard diluent was added to each tube. Following
centrifugation at 1600 g for 30 min at 4°C, radioactivity in the precipitates was measured by a gamma
counter. The sensitivity of this RRA was 0.4 ng/ml.
Gel Chromatography Procedure for Pooled Human
Tears
o
A Sephadex G-50 Superfine (Pharmacia, Piscataway, NJ) column (1.6 X 37 cm) was equilibrated with
1 M acetic acid. Aliquots of tears and urine were
collected from one individual. Two milliliter test
sample or standard hEGF was applied to the column
and eluated at a flow rate of 66 ml/hr. Fractions (0.9
ml each) were collected and lyophilized; hEGF in
each fraction was measured by RIA as described
above.
m
5)
50"
Results
5-
10
20
40
80
160
320
640
1280
hEGF p g / t u b e ( •—• )
Fig. 2. Competitive binding curve of hEGF in tears in radioimmunoassay. Dilution curves generated by tear and urine samples,
from one normal volunteer, and standard hEGF. B/Bo: bound/
total bound ratio.
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Immunoreactive (IR) hEGF was detected in all
human tear samples tested (n = 26). The level of
IR-hEGF in reflex tears (n = 15) varied from 0.7
ng/ml to 8.1 ng/ml, with a mean (±SD) value of 3.4
(±2.9) ng/ml (Fig. 1). IR-hEGF was detectable at similar concentrations (1.9 to 9.7 ng/ml, with a mean
value of 5.3 (±2.9) ng/ml), in non-reflex tears (n
= 11) as well. In contrast, IR-hEGF was undetectable
in aqueous humor by the present RIA system.
1881
EGF IN HUMAN TEARS / Ohoshi er ol
No. 8
Figure 2 shows the standard curve of the hEGF
RIA and the dilution curves of urine and tear samples
obtained from one individual. As demonstrated, the
curves for such samples paralleled the standard curve,
indicating that the activity in the samples measured
by the present RIA system was indistinguishable
from that of standard hEGF. The competitive binding curves of the urine and tear samples in the present
RRA paralleled that for standard hEGF as well (Fig.
3), demonstrating that tear EGF was biologically active. The concentrations of tear EGF estimated by
RIA and RRA were at similar levels: 0.73 ng/ml and
0.68 ng/ml, respectively. Gel filtration study using
Sephadex G-50 Superfine revealed that the IR-hEGF
in tears was eluated as a single peak at the same position as standard hEGF or urine EGF (Fig. 4).
recombinant hEGF
Vo
Vt
I
*
Urine
1-
>
o
(0
<D
O
§ 0.15-1 Tears
E
0.10-1
Discussion
The current study clearly demonstrates that an
EGF which is biologically, immunologically and biochemically indistinguishable from urine EGF or
standard hEGF is present in human tears. The level
of EGF in tears is lower than in urine,10 saliva and
milk," almost comparable to the level in plasma,12
dilution ratio of urine (••
X64
95-,
X32
X16
X8
dilution ratio of tears (AX2
X1
X1/2
50ID
5J
80
160320 640 1280 2560 5120
hEGF pg/tube (•—•)
Fig. 3. Competitive binding curve of hEGF in tears in radioreceptorassay. Dilution curves generated by tear and urine samples,
from one normal volunteer, and standard hEGF. B/Bo: bound/
total bound ratio.
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0.05OJ
30
40
50
60
70
80
fraction No.
Fig. 4. Sephadex G-50 Superfine gel exclusion chromatography
of tear and urine samples from one normal volunteer. Concentration of hEGF was determined by radioimmunoassay. Vo: void
volume, recombinant hEGF: elution volume of l25I-labeled recombinant hEGF. V,: total volume.
and higher than in cerebrospinal fluid13 and pancreatic juice.14 Human tear EGF was shown by RRA to
be biologically active. In contrast to our results, Elliott previously failed to demonstrate EGF immunoreactivity in human tear film.17 This discrepancy may
simply be due to the fact that the present RIA system
is 10 times more sensitive than that used in Elliott's
study.
The above finding is of great importance in view of
the fact that the ocular surface epithelium is always in
contact with tear fluid. Similarly, vitamin A has been
shown to be present in tears as a form of retinol.18
Thus, tear fluid may constitute an environment in
which ocular surface epithelia come into contact with
a variety of nutritional and/or growth factors. It has
been shown that corneal epithelial cells have receptors for EGF19 and proliferate upon topical application of EGF.3'4'6-7 It may be that tear EGF is related to
the maintenance or differentiation of surface epithelia at rest, and to the acceleration of epithelial regeneration after surface wounding. The origin and biological significance of EGF in tear fluid awaits further
investigation.
IR-hEGF was undetectable in human aqueous
humor. Corneal endothelial cells have been shown to
possess EGF receptors20 and to undergo mitosis when
1882
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1989
cultured in the presence of EGF.8 It seems possible,
therefore, that corneal endothelial cells might proliferate when EGF is introduced into the anterior
chamber, although there might be risk of Schlemm's
canal or trabecular cell responses as well.
Key words: epidermal growth factor (EGF), tear, aqueous
humor, human, radioimmunoassay, radioreceptorassay
9.
10.
11.
Acknowledgment
The authors wish to thank Mr. Robert Brady for his
editorial assistance.
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