Glycoprotein methods protocols - biotechnology
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Mucin-Bacterial Binding Assays
Nancy A McNamara, Robert A Sack, and Suzanne M J Fleiszig
1 Introduction
Surface epithelia throughout the body are covered by mucus, a protective secretion that serves as a selective physical barrier between the epithelial cell plasma membrane and the extracellular environment Mucin, the glycoprotein constituent of mucus, has been shown to bind bacteria at mucosal surfaces that line the lung, gut, bladder, oral
cavity, and eye (1–7) Since bacterial binding to an epithelial cell surface is generally thought to be an important prerequisite for infection (8), the interaction between
bac-teria and mucin, together with normal mucosal clearance mechanisms, is believed to act as a defense against infection by inhibiting bacterial adherence to the underlying epithelial cell surface.
In support of mucin’s role as a nonspecific defense mechanism, malfunctions in the production and/or clearance of mucin have been implicated in the etiology of many diseases This has led to the development of methods that can be used to study the effects
of disease and various interventions (e.g., drugs and medical devices) on the ability of mucin to protect the underlying tissue In this chapter, we present methods that can be used to examine the interaction between bacteria and mucin, as well as the extent to which this interaction serves to protect the epithelial cell surface from bacterial invasion.
We have used these methods to study the interactions of Pseudomonas aeruginosa with
the ocular surface in both human and animal models; however, they can also be used to test bacterial/mucin interactions in other tissues with other organisms.
2 Materials
2.1 Preparation and Analysis
of Human Precorneal Tear Film Components
1 Noninvasive corneal irrigation chamber (9,10).
2 0.9% NaCl (autoclaved)
3 Spectra/Por®cellulose ester membranes, 500 Da molecular weight (MW) cut-off (Fisher Scientific, Fair Lawn, NJ)
4 BCA Protein assay kit (Pierce, Rockford, IL)
From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A Corfield © Humana Press Inc., Totowa, NJ
Trang 25 Precast, 4–20% Tris-HCl Ready Gel (Bio-Rad, Hercules, CA) for sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE)
6 Silver stain kit (Bio-Rad)
7 Coomasie blue R-250 (Sigma, St Louis, MO)
2.2 Isolation and Analysis of Human Ocular Sialoglycoprotein
1 Glass microcapillary tubes (calibrated, fire-polished, disposable, 10 µL capacity)
2 SW 4000 size-exclusion column
3 0.5 M NaCl, 0.1 M phosphate buffer, pH 5.0.
4 20% Methanol (v/v)
5 3-kDa Cutoff centrifugal ultrafilters (Filtron, Northborough, MA)
6 Precast, 4% and 4-20% Tris-glycine gels for SDS-PAGE (Novex, San Diego, CA)
7 Kaleidoscope™ (Bio-Rad) and See-Blue ™ (Novex) molecular weight ladders, 6-250 kDa
8 Human lysozyme, IgG, lactoferrin, albumin and sIgA (Sigma)
9 Pre-stained molecular weight markers IgM (990 kDa) and thyroglobulin (669 kDa) (Calbiochem®, La Jolla, CA)
10 Coomasie brilliant blue R-250 (Sigma)
11 Periodate silver and Alcian blue (AB) stains (Sigma)
12 Immobilon P membranes (Millipore, Bedford, MA)
13 Sialyl-Lewis epitope (clone 258-11413, O.E.M Concepts, Toms River, NJ)
2.3 Preparation of Bacteria
1 P aeruginosa (strain 6294, serogroup O6).
2 Trypticase soy agar (TSA) plates (PML Microbiologicals, Wilsonville, OR)
3 Phosphate-buffered saline (PBS), pH 7.4 (Sigma)
4 MacConkey agar plates (PML Microbiologicals)
5 Spectrophotometer
2.4 Microtiter Plate Assay of Bacterial Adherence
1 Linbro/Titertek 96-well microtiter plate (ICN Biomedicals, Aurora, OH)
2 PBS, pH 7.4 (Sigma)
3 0.50% Triton X-100 (LabChem, Pittsburgh, PA)
4 MacConkey agar plates (PML Microbiologicals)
5 Bovine submaxillary gland (BSG) mucin (Sigma)
2.5 Assay to Confirm Adherence of Mucin to Microtiter Wells
1 Same as Subheading 2.4., step 1 (Linbro/Titertek 96-well microtiter plate [ICN
Biomedicals])
2 PBS, pH 7.4 (Sigma)
3 1% Bovine serum albumin (BSA) in PBS (w/v)
4 Biotinylated wheat germ agglutinin (WGA) (Vector, Burlington, CA)
5 PBS/Tween: 0.25 mL Tween-20 (Sigma) in 500 mL PBS
6 PBS/Tween:Streptavidin conjugated alkaline phosphatase (Jackson Immuno Research, West Grove, PN), 500:1 (v/v)
7 Development solution: 5 mM p-nitrophenyl phosphate (NPP) in 0.1 M alkaline buffer
solution (Sigma)
8 Stop solution: 2 M NaCO (2.12 g Na CO/10 mL distilled water)
Trang 32.6 Assay to Measure Bacterial Invasion of Corneal Epithelial Cells
2.6.1 Preparation of Rabbit Corneal Epithelial Cell Cultures
1 Cell culture-treated 96-well plates (Fischer)
2 Modified supplemental hormone essential medium (SHEM) containing 10 µg/mL of
bovine pituitary extract (11).
2.6.2 Gentamicin Survival Assay
1 Buffered minimal essential medium (BMEM): 9.53 g of MEM (Cellgro™) plus 2.2 g of sodium bicarbonate per liter of distilled water (pH to 7.4)
2 Gentamicin sulfate (BioWhittaker, Walkersville, MD)
3 0.25% Triton X-100 (LabChem)
4 BSG mucin (Sigma)
3 Methods
3.1 Preparation and Analysis
of Human Precorneal Tear Film Components
1 Collect precorneal tear film components (TFCs) from the ocular surface of human eyes using a noninvasive corneal irrigation chamber by irrigating each cornea for 30 s with 10 mL of
sterile saline using a metered pump as previously described (9,10).
2 Remove cells and debris by centrifuging the eyewash samples three times at 6000 rpm for
15 min
3 Dialyze the final supernatant against several changes of distilled water at 4°C and con-centrate to 200 µL using vacuum centrifugation
4 Determine the protein content of each 200 µL eyewash sample using the BCA protein
assay kit (12).
5 Separate tear film proteins using a precast 4–20% Tris-HCl gel, and visualize using a silver staining procedure that is ideal for staining polysaccharides and highly glycosylated
proteins as recommended by the manufacturer (Bio-Rad) (13).
6 Counter-stain the silver-stained gels with 200 mL of 0.1% Coomassie brilliant blue R-250 in 25% methanol/7.5% acetic acid for 1 h, destain overnight, and then photograph
in color as previously described (14) (see Note 1).
3.2 Isolation and Analysis of Human Ocular Sialoglycoprotein
1 Collect closed-eye tear samples (which are rich in high molecular weight sialoglycoprotein)
(15) from four human subjects over a period of several weeks as previously described (16).
2 Pool samples and centrifuge at 11,000 rpm in a refrigerated Eppendorf microfuge for
30 min, then repeat Store the resultant supernatants at –70°C until needed
3 Separate the supernatant isocratically in 15-µL aliquots on a SW 4000 size exclusion
column in 0.5 M NaCl, 0.1 M phosphate buffer (pH 5.0), at a flow rate of 0.25 mL/min,
while monitoring the eluent at 254 nm
4 Concentrate each fraction using a 3-kDa centrifugal ultrafilter To establish the elution profile for all the major tear proteins, run fractions under both reducing and non-reducing conditions at 125 V for one hour on a precast, 4–20% Tris-glycine gel for SDS-PAGE Use Kaleidoscope™ and See-Blue™ MW ladders, as well as human lysozyme, IgG, lactoferrin, albumin, and sIgA as standards
5 Using this method, the high molecular weight glycoprotein fractions are recovered slightly after the void volume in the first peak, which elutes off the HPLC column at
Trang 4approxi-mately 24 min To prepare the high molecular weight glycoprotein, collect the initial HPLC fraction, concentrate to 100 µL by centrifugal ultrafiltration, dilute 1:1 with HPLC solvent, and separate into 15-µL aliquots
6 Run high molecular weight glycoprotein fraction under both reducing and nonreducing conditions on a precast 4% Tris-glycine gel with pre-stained molecular weight markers IgM (990 kDa) and thyroglobulin (669 kDa)
7 To detect sialoglycoprotein (SG), periodate treat gel and stain with alcian blue in 3%
acetic acid as previously described (17).
8 For further characterization, transfer glycoprotein overnight onto Immobilon P in 20% methanol (v/v) at 30 V, followed by 80 V for 1 h
9 Probe with a panel of antibodies to specific known mucins, their core proteins, and
com-mon sugar epitopes (see Note 2).
3.3 Preparation of Bacteria for Binding Assay
1 Grow bacteria overnight at 37°C on TSA plates
2 Wash bacteria three times in PBS by centrifugation at 7000 rpm for 5 min (9).
3 Prepare the inoculum by resuspending the washed bacteria into PBS until the optical density at 650 nm reaches 0.1 (equivalent to 1 × 108cfu/mL)
4 Quantify the starting inoculum used in each experiment (typically 1 × 106cfu/mL) by serially diluting the sample and plating 10 µL (in duplicate) on MacConkey agar
3.4 Microtiter Plate Assay of Bacterial Adherence
3.4.1 To Determine Whether or Not Bacteria Bind
to Human Tear Film or Mucin (see Note 3)
1 Coat microtiter wells overnight at 37°C with 100 µL of tear film or mucin sample Pretreat
control wells with PBS which does not promote bacterial adherence to these wells (5,7).
2 Wash wells four times with PBS to remove non-adherent material
3 Prepare bacterial inoculum containing 1 × 106cfu/mL in PBS (10 µL of 1 × 108cfu/mL +
990µL PBS)
4 Add inoculum containing 30 µL of 1 × 106cfu/ml P aeruginosa 6294 to all wells.
5 Incubate plate at 37°C for 30 min
6 Aspirate bacteria with a sterile pipette and wash wells 20 times with PBS to remove nonadherent bacteria
7 Dislodge adherent bacteria from the well surface by adding 300 µL of 0.5% Triton X
8 Incubate plate at 37°C for 30 min
9 Vigorously stir each well with a sterile pipet and perform a viable count by plating 10 µL (in duplicate) on MacConkey agar
3.4.2 To Determine Whether or Not Mucin Blocks Bacterial Adherence
to Known Bacterial Binding Factors (see Note 4)
1 Coat microtiter wells overnight at 37°C with 100 µL of known bacterial binding factor (e.g., TFCs or BSG mucin)
2 Wash wells four times with PBS to remove nonadherent material
3 Coat microtiter wells with 100 µL of SG for 18 h at 37°C Treat control wells with 100 µL of PBS instead of SG since PBS does not affect bacterial binding to either TFCs or BSG mucin
4 Prepare bacterial inoculum containing 1 × 105 cfu/mL in PBS (1 µL of 1 × 108cfu/mL +
999µL PBS)
5 Add inoculum containing 30 µL of 1 × 105cfu/mL P aeruginosa 6294 to all wells.
Trang 56 Incubate plate at 37°C for 30 min.
7 Wash wells 20 times with PBS to remove nonadherent bacteria
8 Dislodge adherent bacteria from the well surface by adding 300 µL of 0.5% Triton X
9 Incubate plate at 37°C for 30 min
10 Vigorously stir each well with a sterile pipet and perform a viable count by plating 10 µL (in duplicate) on MacConkey agar
3.4.3 To Determine Whether or Not Treating Bacteria with Mucin Blocks Their Ability to Adhere to Known Bacterial Binding Factors (see Note 4)
1 Coat microtiter wells overnight at 37°C with 100 µL of known bacterial binding factor (e.g., TFCs or BSG mucin)
2 Wash wells four times with PBS to remove nonadherent material
3 Prepare bacterial inoculum containing 2 × 105 cfu/mL in PBS (2 µL of 1 × 108cfu/mL +
998µLl PBS)
4 Prepare starting inoculum by mixing 100 µL of 2 × 105cfu/mLP aeruginosa (prepared
above) with 100 µL of SG and incubate at 37°C for 1 h Prepare inoculum for controls by mixing 100 µL of 2 × 105cfu/mL bacteria with 100 µL of PBS (see Note 5).
5 Add 30 µL of the starting inoculum containing P aeruginosa 6294 (1 × 105cfu/mL) and either SG or PBS (control) to wells
6 Incubate plate at 37°C for 30 min
7 Wash wells 20 times with PBS to remove nonadherent bacteria
8 Dislodge adherent bacteria from the well surface by adding 300 µL of 0.5% Triton X
9 Incubate plate at 37°C for 30 minu
10 Vigorously stir each well with a sterile pipet and perform a viable count by plating 10 µL (in duplicate) on MacConkey agar
3.5 Assay to Confirm Adherence of Mucin to Microtiter Wells
1 Coat 96-well microtiter plate overnight at 37°C with several dilutions of SG, TFC, and BSG mucin
2 Wash wells 24 times with PBS
3 Block for 2 h at room temperature (or overnight at 4°C) by adding 200 µL of 1% BSA/ PBS to each well
4 After blocking, wash wells twice with PBS/Tween and incubate with 100 µL of 5 µg/mL biotinylated wheat germ agglutinin (WGA) for 45 min at room temperature (wrap plate in plas-tic) WGA is a plant lectin that binds specifically to sialic acid residues, and thus, serves as a probe for quantifying the amount of sialylated glycoprotein that is bound to the microtiter well
5 Wash wells six times with PBS/Tween
6 Detect WGA-bound biotin by adding 100 µL of PBS/Tween:streptavidin-peroxidase solution to each well and incubating the wrapped plate at 37°C for 45 min
7 Wash wells six times with PBS/Tween
8 Add 100 µL of development solution (NPP in alkaline buffer) and incubate at room tem-perature Detect the sialoglycoprotein-lectin-enzyme complexes by adding the enzyme sub-strate (NPP) which is converted to a colored product in the presence of enzyme Immediately begin monitoring the intensity at 405 nm using a standard ELISA reader Readings should
be taken every 2 min since development occurs quickly Controls consist of ovalbumin
treated wells incubated with the WGA and/or streptavidin system alone (18,19).
9 Stop the reaction by adding 10 µL of stop solution This step can be omitted if the absorbance
is monitored continuously following the addition of development solution (see Note 6).
Trang 63.6 Assay to Measure Bacterial Invasion of Corneal Epithelial Cells ( see Note 7)
3.6.1 Preparation of Rabbit Corneal Epithelial Cell Cultures
1 Place 100 µL of SHEM suspension containing rabbit corneal epithelial cells in cell cul-ture-treated 96-well plates
2 Maintain cells at 37°C while changing SHEM every 2 d until confluent (typically 4–7 d)
3.6.2 Preparation of Bacteria for Gentamicin Survival Assay
( see Subheading 2.3 for Materials)
1 Grow bacteria overnight at 37°C on TSA
2 Prepare the inoculum by resuspending bacteria in 5 mL of buffered Eagle’s minimal essential medium (BMEM) using a sterile cotton swab until the optical density reaches 0.1 (equivalent to 1 × 108cfu/mL at 650 nm)
3 The starting inoculum of 1 × 107cfu/mL is prepared by placing 100 µL of the bacterial suspension into 900 µL of BMEM Quantify the starting inoculum used in each experi-ment by serially diluting the sample and plating 10 µL (in duplicate) on MacConkey agar 3.6.3 Gentamicin Survival Assay
1 Wash confluent cells by adding 200 µL of BMEM to each well and aspirating with a sterile pipet
2 Coat cells with 70 µL of BMEM containing several concentrations of BSG mucin, TFC,
or SG Pretreat control cells with 70 µL of BMEM alone
3 Incubate plate at 37°C for 2 h
4 Add 8 µL of an inoculum containing 1 × 107 cfu/mL of P aeruginosa 6294 to all wells.
5 Incubate plate at 37°C for 3 h
6 Perform viable counts on each well after 3 h to estimate the number of bacteria in the
extracellular medium (see Note 8).
7 Aspirate remaining medium and kill extracellular bacteria by adding 200 µL gentamicin solution (200 µg/mL) to each well
8 Incubate plate at 37°C for 2 h
9 Wash cells with 200 µL BMEM
10 Lyse cells with 100 µL 0.25% Triton-X solution
11 After 15 min, use a sterile pipet to vigorously stir each well Perform a viable count to quantify intracellular bacteria by plating 10 µL (in duplicate) on MacConkey agar
Com-pare the susceptibility of cells to invasion by P aeruginosa in the presence of mucin with
that of control cells incubated with medium alone (see Note 9).
4 Notes
1 Using the silver/coomassie blue double-staining method described in Subheading 3.1.,
all of the silver-stained glycoproteins and lipids stain by silver only and appear dark gray, whereas all of the unglycosylated proteins stain with coomassie blue and appear blue Thus, polypeptides stained by coomassie brilliant blue and glycosylated proteins stained
by silver can be differentially visualized in the same gel With this method, whole human tear film samples are shown to contain a prominent high molecular weight silver-stained band of glycosylated protein (>200 kDa) that does not stain with coomassie brilliant blue Others have identified a glycoprotein of similar size in human tear samples collected with
a microcapillary tube (20–22).
Trang 72 SDS-PAGE in combination with periodate silver and AB staining (described in Sub-heading 3.2.) reveal that high molecuar weight glycoproteins isolated from the ocular
surface consist primarily of three SG bands, one in the stacking gel and two in the running gel with apparent molecuar weight ranges of 500–450 kDa In addition, all of the SGs isolated from human tear film exhibit a common reactivity with a mAb raised against a sialyl-Lewis epitope specific to salivary mucin (clone 258–11413, O.E.M Concepts) Immunofluorescence microscopy reveals intense specific staining of the conjunctival epithelial plasma membrane but not the goblet cells on probing with the mAb to salivary
mucin, suggesting that these SGs arise specifically from the epithelium (15).
3 In a previous study we showed that P aeruginosa binds to factors present in whole human
tears (23) To examine whether the bacterial binding factors in precorneal human tear
film may include mucin, we used the bacterial adherence assay, described in Subheading 3.4., and compared bacterial adherence to the SG isolated from the human ocular surface
to binding elicited with whole human TFCs and BSG mucin
4 Using the bacterial adherence assay, we found that P aeruginosa adhered to wells coated
with both whole human tear film and BSG mucin, but did not bind to wells coated with either ocular SG or PBS alone This suggested that human ocular SG was not responsible for bacterial binding to whole tear film and that the bacterial binding factor(s) in human tears are either other glycoproteins or nonglycoprotein fractions Having ruled out bacterial binding to SG, we developed two new methods, which were variations of the original bacte-rial adherence assay, to determining whether SG could inhibit bactebacte-rial interaction with known bacterial binding factors The first variation of the bacterial adherence assay, described in
Subheading 3.4.2., sought to determine whether or not ocular SG could block bacterial
bind-ing to whole human tear film or to BSG mucin when microtiter plates were precoated with TFCs or mucin and then subsequently coated with SG The second variation, described in
Subheading 3.4.3., was used to determined whether or not bacterial binding to mucin-coated
microtiter plates could be blocked by adding SG to the bacterial inoculum
5 When bacteria are pretreated with any factor prior to performing the bacterial adherence assay, one must demonstrate that preincubation with that factor does not reduce the viability or the replication rate of the bacteria This can be accomplished by performing viable counts after 2 and 8 h to determine whether inocula containing treated and untreated bacteria are equivalent
6 Using the biotinylated WGA assay described in Subheading 3.6., we found that the
absorbance reading for a 10–2dilution of SG was similar to that observed for a 10–2 dilu-tion of TFCs and 10 µg/mL of BSG mucin; thus, we used these concentrations in the bacterial binding assay to ensure that an equivalent number of sialic acid residues were bound to the bottom of the well for each preparation
7 Bacterial association with the corneal surface is a crucial step towards bacterial coloniza-tion and invasion of corneal epithelial cells Since soluble factors in whole human tears
and BSG mucin bind P aeruginosa, while human ocular SG does not, we used the gen-tamicin survival assay to examine whether these factors could block P aeruginosa
inva-sion of corneal epithelial cells As was expected, treating cells with several different dilutions of BSG mucin or TFCs caused a two- to threefold decrease in susceptibility to
bacterial invasion by P aeruginosa strain 6294, whereas ocular SG did not significantly
enhance or inhibit bacterial invasion of corneal epithelial cells compared to controls
8 When studying bacterial invasion of epithelial cells using the gentamicin survival assay,
it is important to confirm that an equivalent number of extracellular bacteria remain in the wells following each treatment (i.e., the treatment does not alter the viability or
Trang 8replica-tion rate of the bacteria) This is best done by performing a viable count on one well in each different treatment group at the end of the 3-h incubation of cells with bacteria (just prior to adding the gentamicin)
9 For all the assays described in this chapter, we recommend that at least six wells are used for each sample group in each experiment and that experiments are repeated a minimum
of three times Nonparametric statistical techniques are typically used to make compari-sons since the presence of outlying values may cause distributions to deviate from nor-mality Two group comparisons can be made with the Wilcoxan 2-group test for unpaired data and the Kruskal-Wallis test can be used for the comparison of three or more indepen-dent groups of data
References
1 Florey, H (1955) Mucin and the protection of the body Proc R Soc Lond 143, 147–158.
2 Liljemark, W and Gibbons, R (1972) Proportional distribution and relative adherence of
Streptococcus miteor (mitis) on various surfaces in the human oral cavity Infect Immun.
6, 852–859.
3 Parsons, C and Mulholland, S (1978) Bladder surface mucin Its antibacterial effect
against various bacterial species Am J Pathol 93, 423–432.
4 Cohen, R., Rossoll, R., Cabelli, V., Yang, S., and Laux, D (1983) Relationship between
the mouse colonizing ability of a human fecal Escherichia coli strain and its ability to bind
a specific mouse colonic mucous gel protein Infect Immun 40, 62–69.
5 Vishwanath, S and Ramphal, R (1984) Adherence of Pseudomonas aeruginosa to human
tracheobronchial mucin Infect Immunity 45, 197–202.
6 Chick, S., Harber, M., Mackenzie, R., and Asscher, A (1981) Modified method for studying
bacterial adhesion to isolated uroepithelial cells and uromucoid Infect Immun 34, 256–261.
7 Fleiszig, S., Zaidi, T., Ramphal, R., and Pier, G (1994) Modulation of Pseudomonas
aeruginosa adherence to the corneal surface by mucus Infect Immun 62, 1799–1804.
8 Ramphal, R., McNiece, M., and Polack, F (1981) Adherence of Pseudomonas aeruginosa
to the injured cornea A step in the pathogenesis of corneal infections Ann Ophthalmol.
100, 1956–1958.
9 Fleiszig, S., Efron, N., and Pier, G (1992) Extended contact lens wear enhances
Pseudomonas aeruginosa adherence to human corneal epithelium Invest Ophthalmol.
Vis Sci 33, 2908–2916.
10 Fullard, R and Wilson, G (1986) Investigation of sloughed corneal epithelial cells
col-lected by non-invasive irrigation of the corneal surface Curr Eye Res 5, 847–856.
11 Jumblatt, M and Neugeld, A (1983) Serotonergic responsiveness of rabbit corneal
epi-thelial cells in culture Invest Ophthalmol Vis.Sci 24, 1139–1143.
12 Lowry, O., Rosebrough, N., Farr, A., and Randall, R (1951) Protein measurement with
the folin phenol reagent J Biol Chem 193, 265–275.
13 Merril, C., Goldman, D., Sedman, S., and Ebert, M (1981) Ultrasensitive stain for
pro-teins in polyacrylamide gels show regional variations in cerebrospinal fluid propro-teins
Sci-ence 211, 1437.
14 Dzandu, J., Deh, M., Varratt, D., and Wise G (1984) Detection of erythrocyte membrane proteins, sialoglycoproteins and lipids in the same polyacrylamide gel using a double
stain-ing technique Proc Natl Acad Sci USA 81, 1733–1737.
15 Sack, R., Bogart, B., Beaton, A., Sathe, S., and Lew, G (1997) Diurnal variations in tear glycoproteins Evidence for an epithelial origin for the major non-reducible ≥ 450 kDa
sialoglycoprotein(s) Curr Eye Res 16, 577–588.
Trang 916 Sack, R., Tan, K., and Tan, A (1992) Diurnal tear cycle: evidence for a nocturnal
inflam-matory constitutive tear fluid Invest Ophthalmol Vis Sci 33, 626–640.
17 Wardi, A and Michos, G (1972) Alcian blue staining of glycoproteins in acrylamide disc
electrophoresis Anal Biochem 49, 607–609.
18 Kaku, H., Mori, Y., Goldstein, I., and Shibuya, N (1993) Monomeric, monovalent
deriva-tive of Maackia amurensis leukoagglutinin J Biol Chem 268, 13,237–13,241.
19 Rogerieux, R., Belaise, M., Terzidis-Trabelsi, H., Greffard, A., Pilatte, Y., and Lambre, C (1993) Determination of the sialic acid linkage specificity of sialidases using lectins in a
solid phase assay Anal Biochem 211, 200–204.
20 Moore, J and Tiffany, J (1979) Human ocular mucus Origins and preliminary
character-ization Exp Eye Res 29, 291–301.
21 Chao, C., Vergnes, J., and Brown, S (1983) O-glycosidic linkage in glycoprotein isolates
from human ocular mucus Exp Eye Res 37, 533–541.
22 Jay, G and Hong, B (1992) Characterization of a bovine synovial fluid lubricating factor
II Comparison with purified ocular and salivary mucin Conn Tissue Res 28, 89–98.