Experimental and Molecular Pathology 79 (2005) 206 – 209

www.elsevier.com/locate/yexmp

Comparison of antibody array substrates and the use of glycerol

to normalize spot morphology
Eric W. Olle a,c, James Messamore b, Michael P. Deogracias c, Shannon D. McClintock c,
Timothy D. Anderson a, Kent J. Johnson c,*
a
Pfizer Global Research and Development, Safety Sciences, Ann Arbor, MI 48105, USA
b
Pfizer Global Research and Development, Discovery-Biomarkers Group, Ann Arbor, MI 48105, USA
c
Department of Pathology, The University of Michigan School of Medicine, 1301 Catherine Rd., Ann Arbor, MI 48109-0602, USA

Received 27 August 2005

Available online 24 October 2005

Abstract

Antibody microarrays are a high-throughput proteomic technology used to examine the expression of multiple proteins in complex solutions.
Antibody microarrays can be manufactured on a variety of commercially available activated glass or coated slides. The goal of this study was to
compare Hydrogeli, nitrocellulose, aldehyde-silane and epoxy-silane slides to determine the amount of antibody bound. The optimal substrate
was defined as one that bound the greatest amount of antibody with minimal background. Our studies found that epoxy-silane enhanced surface
(ES) slides gave the greatest degree of binding along with a minimal background. However, larger antibody microarrays showed variability in spot
size, high intra-spot coefficient of variation and drying artifacts. Increasing the amount of glycerol in the spotting buffer caused a dose-dependent
improvement in overall spot morphology. Glycerol was tested on 128 different antibodies and showed decreased: mean spot diameter, intra-spot
coefficient of variation and drying artifacts. These studies revealed that the optimal slide substrate was epoxy-silane ES microarray slides.
Furthermore, glycerol could normalize spot size, decrease intra-spot coefficient of variability, decrease drying artifacts and increase antibody-
spotting density.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Antibody microarray; Antibody spot morphology; Buffer optimization

Introduction commonly used are nitrocellulose, hydrogel or activated glass

Antibody arrays are a protein microarray technology that
allow researchers to examine the expression of a large number
of proteins simultaneously (Haab et al., 2001; Schweitzer et al.,
2002; Sreekumar and Chinnaiyan, 2002a,b; Sreekumar et al.,
2001). The antibodies are immobilized on a range of different
substrates or coated slides including poly-l-lysine (Haab et al.,
2001; Neuman de Vegvar et al., 2003; Sreekumar and
Chinnaiyan, 2002a), nitrocellulose (Huang et al., 2001; Knight
et al., 2004), hydrogel/polyacrylamide gel (Rubina et al., 2003;
Zhou et al., 2004), agarose (Afanassiev et al., 2000), aldehyde
(MacBeath and Schreiber, 2000), epoxy (Letarte et al., 2005;
Seong, 2002) or other polymeric coatings (Angenendt et al.,
2003; Cretich et al., 2004; Oh et al., 2005). The substrate types
* Corresponding author. Fax: +1 734 764 4308.
E-mail address: kjjkjj@umich.edu (K.J. Johnson).
0014-4800/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexmp.2005.09.003
slides (i.e. epoxy or aldehyde) (Angenendt et al., 2002, 2003;
Kusnezow and Hoheisel, 2003; Seong, 2002; Wu and Grainger,
2004). Nitrocellulose membranes (Huang et al., 2001),
nitrocellulose slides (Knight et al., 2004) and hydrogel coated
slides (Zhou et al., 2004) allow for the use of multiple buffer
types without pH modification or the need to use tertiary amine
free buffers. Activated glass slides from DNA microarray
technology are starting to receive additional attention in the
manufacture of protein microarrays (Angenendt et al., 2002;
Seong, 2002). The use of epoxy-silane or aldehyde-silane
allows for covalent linkages to tertiary amine groups on amino
acids. Alkaline buffers enhance the covalent attachment of the
antibodies to activated glass slides (Seong, 2002). However,
application of the DNA microarray slide technology to
antibody microarrays requires selection and optimization of
the slide (Kusnezow and Hoheisel, 2003; Seong, 2002; Seong
and Choi, 2003).
 E.W. Olle et al. / Experimental and Molecular Pathology 79 (2005) 206 – 209
The goal of this study was to analyze commonly used slide
substrates to determine the optimal type for antibody micro-
arrays. Selection criteria were based on a high signal to noise
ratio when binding a biotin-conjugated antibody to the slide.
Once a slide substrate with good antibody binding character-
istics was identified, the spotting buffer was modified to provide
minimal antibody-to-antibody spot size differences and low
intra-spot variability.
Methods
Different slide substrates were compared by spotting a biotinylated anti-goat
IgG antibody (R&D, Minneapolis, MN) at a concentration of 25 Ag/ml onto:
Nitrocellulose Fast slides (Schleicher and Schuell BioScience Inc., Keene, NH),
hydrogel (Perkin-Elmer, Boston, MA), ArrayIti SuperAldehyde (Telechem,
Sunnyvale, CA), epoxy-silane or epoxy-silane ES (Erie Scientific, Portsmouth,
NH). Antibodies spotted on nitrocellulose or hydrogel slides were diluted in
phosphate-buffered saline (PBS) (Gibco/Invitrogen, Carlsbad, CA) with 50 mM
trehalose (Sigma Chemical Co, St. Louis, MO). Antibodies spotted on activated
epoxy or aldehyde glass slides were diluted in 100 mM NaHPO4 pH 9.0, 50 mM
NaCl and 50 mM trehalose. Diluted antibodies were spotted using a Piezorray
non-contact printing system (Perkin-Elmer) following manufacturer’s instruc-
tions. Spotting occurred at 45 – 50% humidity with the slides cooled to 2-C
above T d (dew point). Slides were stored for 2 days at 4-C in a desiccated
environment. Slides were washed 3  5 min in TBS with 0.5% Tween-20 (TBS-
t) (Sigma) by placing a single slide in a 50 ml tube on a vertically rotating
platform. The antibody was detected using streptavidin Cy3 (Zymed, South San
Francisco, CA) diluted 1:5000 in TBS-t with 1% dry milk placed into a heat-
sealed packet. The packet was taped onto a vertically rotating platform for 30
min at room temperature. The slides were removed from the packet and dipped
in TBS-t to remove excess detection buffer and washed as above. Slides were
dipped in 18 mV water and dried using compressed air. Slides were scanned on
an Axon 4000B scanner (Axon Instruments Inc., Union City, CA), and spot
intensity and background were determined using GenePix Pro 4.0 (Axon).
Analysis of different antibodies on antibody microarrays followed the
protocol outlined above except, after the initial wash, the arrays were
hybridized with biotin-conjugated universal secondary antibody solution
(Phoenix Biotechnologies, Huntsville, AL) supplemented with biotin-conju-
gated donkey anti-goat IgG (Chemicon, Temecula, CA) in a pouch for 1
h followed by 3  5 min washes in TBS-t. The antibodies were detected,
scanned and analyzed as above. Data were exported and analyzed with a two-
tailed t test using PrismGraph 4.0 (GraphPad Software, Inc. San Diego, CA).
Results
Comparing the binding of IgG to various slide substrates
indicated that there were differences in the spot fluorescence
intensity and diameter. The epoxy-silane ES slides have a
greater median fluorescence signal than hydrogel and other
activated glass slides (Table 1). Background fluorescence
intensity was similar on the hydrogel and activated glass slides,
with epoxy-silane having the least. Nitrocellulose-based slides
were tested but excluded due to a low signal to noise ratio with
these test conditions (data not shown). Thus, the epoxy-silane
ES slides appeared to have the optimal degree of antibody
binding, and further experiments used these slides.
In an attempt to standardize the spot size, glycerol was
added to the spotting buffer at 0.5%, 1.0%, 2.5% and 5% to test
its effect on the Piezorray spot volume and spot size on epoxy-
silane ES-coated slides. The effect of glycerol on Piezorray
spot volume was measured using the ‘‘tip tuning utility’’
(Piezorray software) and showed that glycerol had minimal
207
Table 1
Comparisons of different types of antibody array slide substrates
Slide type Median Background Mean spot
fluorescence fluorescence diameter (Am)*
Hydrogeli 17,320 T 1103 502 T 22 170
(Perkin-Elmer)
SuperAldehydei 11,101 T 693 483 T 19 140
(Telechem)
Epoxy-silane 14,321 T 701 407 T 21 130
(Erie Scientific)
Epoxy-silane ES 20,931 T 1182 474 T 17 120
(Erie Scientific)
Small antibody microarrays were printed using the Piezorray non-contact
system with biotinylated anti-goat IgG antibody (R&D) at a concentration of 25
Ag/ml. The median fluorescent intensity of the antibody spot, surrounding
background and spot diameter (10 AM resolution) were measured on an Axon
4000B scanner. * = mean spot diameter without the addition of glycerol.
Antibody microarrays were composed of 128 spots with the spotting and
hybridization repeated 4 times.
effect of spot volume. Up to 5%, glycerol had no effect on
Piezorray spot volume (Fig. 1A).
Two antibodies that were found to produce significantly
different spot sizes with the same protein concentration were an
anti-IL-1h (small spots) and a mouse IgG2h isotype control
antibody (large spots) (R&D). A small antibody array containing
anti-IL-1h and mouse IgG2h isotype control was used to
determine if glycerol could help normalize the spot diameter.
Glycerol appeared to decrease the spot size of the isotype control
antibody with a concurrent increase in anti-IL-1h spot size (Fig.
1B). Statistical comparison of the increasing amounts of glycerol
on epoxy-silane ES slides showed that there were statistical
differences ( P < 0.001) between the spot size of anti-IL-1h and
the isotype control antibodies in all groups except for 5%
glycerol (Fig. 1B). Increasing the amount of glycerol in the
spotting buffer had an added benefit of decreasing the intra-spot
coefficient of variation (Fig. 1C). Finally, a larger array
consisting of 128 different antibodies from a range of suppliers
spotted in sextuplicate showed that 5% glycerol showed a
decrease in the mean size and when compared to 0.5% glycerol
(t test P < 0.0001) (Fig. 1D). Glycerol also decreased the
variability of spot diameter.
The effect of varying the concentrations of glycerol can also
be applied to the standard epoxy-silane and possibly all
activated glass slides that lack the roughened ES surface. The
epoxy-silane ES and epoxy-silane slides were spotted using the
increasing amount of glycerol, and, as shown in Fig. 2, there
was normalization of spot size and CV with both substrates,
with 5% glycerol being the most effective. Thus, in addition to
the epoxy-silane ES slides, the use of glycerol to control spot
morphology worked effectively in the standard epoxy-silane
and aldehyde (data not shown) slides and could be applied to
decrease drying artifacts.
Discussion
Despite the use of activated glass slides in DNA
microarrays (Angenendt et al., 2002; MacBeath and Schrei-
208 E.W. Olle et al. / Experimental and Molecular Pathology 79 (2005) 206 – 209

Fig. 1. Effect of glycerol on antibody spot volume, size distribution and morphology. The effect of 5% glycerol on spot volume was tested in the Piezorray tip tuning
utility (A). Control spotting liquid was PBS that was compared to the spotting buffer with 5% glycerol. Bar graph shows mean T standard deviation n = 10. Epoxy-
silane ES slides were analyzed for spot diameter. Graph (B) shows mean spot diameter of isotype control and anti-IL-1h antibodies spotted 128 times from 4
independent experiments, with error bars showing standard deviation, and statistically significant differences in spot size are indicated by *** (t test P < 0.001). The
effect of glycerol on intra-spot variability was plotted showing the mean coefficient of variation (C). A large array containing 128 different antibodies spotted in
sextuplicate were printed using either 0.5% or 5% glycerol (n = 5). Spot diameter was analyzed and plotted using a whisker plot (D). Statistical comparison on the
two groups using a two-tailed unpaired t test showed a difference between the two groups indicated by **** (P < 0.0001).

ber, 2000; Seong, 2002), the use of epoxy-silane-coated

Fig. 2. Effect of glycerol on antibody spot morphology. Representative sub-array
spotted on either epoxy-silane ES or epoxy-silane using different amounts of
glycerol to compare the standard with the roughened surface of the ES slides.
Antibodies were detected using biotin-conjugated universal secondary antibody
and Cy3 streptavidin. Arrays were scanned using Axon 4000B scanner and
GenePix Pro 4.0.
slides for antibody microarrays is limited to a few references
(Letarte et al., 2005; Olle et al., in press; Seong, 2002).
While the majority of antibody arrays use hydrogel- or
nitrocellulose-coated slides, epoxy-silane-coated ES slides
have a greater median fluorescence with lower background
than hydrogel. Nitrocellulose slides were tested but excluded
from the experiment due to requiring 4- to 10-fold more
antibody than glass/hydrogel-based slides and having a high
non-specific background. Thus, our studies show that epoxy-
silane ES slides were the best substrate to use for signal
intensity, spot diameter and low background.
The fact that epoxy-silane ES slides had a high signal to
noise ratio is necessary for developing antibody microarrays
that are reproducible and have a low limit of detection. The
lower non-specific background allows for a theoretical
lower limit of detection, and the smaller spot diameter
allows for greater array density. One problem encountered
as antibody microarrays grew larger was variability in the
spot diameter, which made it difficult to effectively use
array space. Another problem overcame with the glycerol
 E.W. Olle et al. / Experimental and Molecular Pathology 79 (2005) 206 – 209
was intra-spot variability and drying artifacts. Glycerol-nor-
malized spot size causing increased spotting density decreases
intra-spot signal variability and slide-to-slide variability. When
comparing multiple antibodies, the glycerol concentration was
set at the low end at 0.5% to account for some of the antibodies
stored in 50% glycerol. When the 0.5% was compared to 5%
glycerol across 128 different antibodies, the overall spot
diameter and variability decreased. The suitability of epoxy-
silane microarray slides has been demonstrated before, but these
studies did not optimize antibody spot variability across
multiple antibodies from multiple vendors and sources (Letarte
et al., 2005; Seong, 2002).
The effect of glycerol on spot morphology and coefficient of
variation was greater in the standard epoxy-silane slides. This is
due to the slightly roughened surface on the epoxy-silane ES
slides that increase epoxy group density and decrease spreading.
The addition of glycerol normalized and caused an overall
reduction in the mean spot diameter. One problem with the
smaller spot diameter is that the increased antibody-spotting
density may cause non-specific binding, however, this has not
been a problem when the capture antibody concentration is
titrated. As a control for non-specific antibody– antigen inter-
actions, isotype controls at the same spotting density should be
included. Since antibody spot diameters are similar, it allows for
direct comparison of the analyte and the isotype background
controls.
Epoxy-silane or aldehyde-silane linkages are enhanced at an
alkaline pH, and tertiary amine containing buffers should be
avoided since the compete for the slides active groups. We have
seen no evidence of decreased antibody activity by using
alkaline spotting. Antibodies stored or reconstituted in tertiary
amine containing buffers can be dialyzed into phosphate-
buffered saline without incident in the majority of cases.
In summary, epoxy-silane-modified glass slides are an
effective substrate for the application of antibodies and can be
used in conjunction with glycerol to normalize the spot
variability and spot size. Epoxy-silane slides have the added
benefit of requiring less capture antibody when compared with
Hydrogeli and nitrocellulose slides (Huang et al., 2001;
Knight et al., 2004; Zhou et al., 2004).
Acknowledgments
The authors wish to thank Beverly Schumann for her help
with the preparation of the manuscript and Dr. Tiffany M.
Lasky for her evaluation of the manuscript.
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