Journal of Immunological Methods 255 Ž2001. 1–13
Detection of multiple proteins in an antibody-based protein
Ruo-Pan Huang )
DiÕision of Research, Department of Gynecology and Obstetrics, Emory UniÕersity School of Medicine, 1639 Pierce DriÕe, Room 4219,
Atlanta, GA 30322, USA
Received 9 January 2001; received in revised form 27 March 2001; accepted 28 March 2001
A new antibody-based protein array assay is described. This assay combines the advantages of the specificity of
enzyme-linked immunosorbent assays ŽELISA., sensitivity of enhanced chemiluminescence ŽECL. and high-throughput of
microspot. In this system, the capture proteins, either antibodies or antigens are spotted onto membranes in an array format.
Biological samples are then incubated with membranes. After antigens or antibodies in the samples bind to their
corresponding targets and unbound proteins are washed away, the membranes are exposed to Horseradish Peroxidase
ŽHRP.-conjugated antibodyŽies.. The signals are finally visualized with ECL system. Experiments demonstrate that multiple
cytokines and antibodies can be simultaneously detected using this new approach. The procedure is so simple that no
sophisticated equipment is required. The concept should be able to be extended to develop a high-throughput protein array
system. Future applications of this new approach include direct protein expression profiling, immunological disease
diagnostics and discovery of new biomarkers. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Protein arrays; ELISA; Cytokine; Antibody; Protein expression
1. Introduction genomic research alone. Although DNA is an infor-
mation archive, proteins do almost all the work of
With the complete sequence of human genome, the cell. Experimental evidence clearly shows a dis-
we now should be able to analyze whole genomic parity between the relative expression levels of
expression. This will provide vital information on the mRNA and their corresponding proteins ŽGygi et al.,
coordinate regulation among many genes since al- 1999.. More importantly, the protein-based analysis
most all cell activities or phenotypes are the sum of a is able to study post-transcriptional control, post-
series of molecular and biochemical events interact- translational modifications and protein–protein inter-
ing with each other in a complex and multifaceted action. Therefore, the field of proteomics with an
fashion. However, a complete understanding of nor- ultimate goal to assemble a complete library of all
mal and disease states can never be obtained from the proteins is becoming increasingly important
ŽAnderson et al., 2000; Legrain et al., 2000.. Unlike
Tel.: q1-404-712-9673; fax: q1-404-727-8615.
the cDNA microarray technology, the methodology
E-mail addresses: firstname.lastname@example.org, email@example.com that allows detecting entire pool of proteins does not
ŽR.-P. Huang.. exist yet. Two systems, two-dimensional polyacy-
0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 3 9 4 - 5
2 R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13
lamide gel electrophoresis Ž2-D gel. coupled with oratories ŽWest Grove, PA.. HRP-conjugated anti-
mass spectrometry ŽEmmert-Buck et al., 2000; Page donkey IgG was purchased from Rockland ŽGilberts-
et al., 1999; Haynes and Yates, 2000; Celis et al., ville, PA.. Agarose immobilized guinea pig IgG,
1999. and surface-enhanced laser desorption and ion- goat IgG, human IgG and sheep IgG were also
ization ŽSELDI. ŽKuwata et al., 1998; Bruenner et purchased from Rockland.
al., 1996. are currently being used in analysis of Membranes were provided by several companies
multiple protein expression. However, the require- as indicated in Table 2.
ment of sophisticated devices greatly limits their
accessibility. In this study, I demonstrated that nu- 2.1. Preparation of array membranes
merous proteins could be detected simultaneously
and specifically using an ELISA-based protein array A template of 504 spots with 28 spots in width
system. This method can be used to detect multiple and 18 spots in length in a size of 6 = 8 cm was
secreted proteins and antibodies. generated from a computer. This template was used
as a guide to spot solution onto membranes. To spot
capture proteins onto membranes, the template was
2. Materials and methods placed on the top of white light box. Membranes
were then put on the top of the template. Through
Immunoglobulins ŽIgGs. and corresponding the light, dark spots in the template were clearly
Horseradish Peroxidase ŽHRP.-conjugated mono- visible from the membrane and used to guide to spot
clonal antibodies were purchased from several differ- solution onto membranes. 0.25 ml of solution was
ent companies as shown in Table 1. IgGs were manually loaded onto a single spot by a 2-ml pipet-
prepared as stock solutions at a concentration of 4 man. Typically it took about 30 to 50 min to spot the
mgrml and diluted with TBS to 100 mgrml as entire membrane. HRP-conjugated or biotin-con-
working solutions prior to experiments. jugated antibodies were spotted onto membranes as
Pairs of antibodies against cytokines were ob- positive control and identification of orientation of
tained from BD PharMingen ŽSan Diego, CA.. Cy- arrays.
tokines were purchased from Peprotech ŽRochy Hill,
NJ.. Cytokines were prepared as stock solutions at a 2.2. Array assay of different species of HRP-con-
concentration of 100 mgrml and diluted into suit- jugated IgGs
able working concentrations prior to experiments.
All of donkey anti-Igs against specific species Different IgGs Ž0.25 ml of 100 mgrml. were
were purchased from Jackson ImmunoResearch Lab- loaded onto membranes as described above. Mem-
branes were blocked with 5% BSA ŽBovine Serum
Albumin.rTBS Ž0.01 M Tris HCl pH 7.6r0.15 M
Table 1 NaCl. for 1 h at room temperature and incubated
Array antigens and corresponding antibodies individually or collectively with HRP-conjugated an-
JIRL: Jackson ImmunoResearch Laboratories; SCB: Santa Cruz
tibodies for 2 h at room temperature. Arrays were
then washed three times with TBSr0.1% Tween 20
Antigens Sources Antibodies Sources
and then twice with TBS. The signals were finally
Bovine IgG Sigma Anti-bovine IgG JIRL imaged with enhanced chemiluminescence ŽECL..
Chicken IgG Sigma Anti-chicken IgG JIRL
Goat IgG Sigma Anti-goat IgG SCB
Guinea pig IgG Sigma Anti-guinea pig IgG JIRL 2.3. Array assay of multiple cytokines
Human IgG Sigma Anti-human IgG JIRL
Mouse IgG Sigma Anti-mouse IgG Sigma A pair of antibodies that recognize different epi-
Rabbit IgG Sigma Anti-rabbit IgG Sigma topes of same antigen was used to capture and detect
Rat IgG Sigma Anti-rat IgG Amersham
Sheep IgG Sigma Anti-sheep IgG JIRL
a certain antigen. 0.25 ml of individual capture anti-
BSA Roche body at a concentration of 200 mgrml was spotted
onto membranes as described above. After blocking
R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13 3
Membranes for protein arrays
MSI: Micron separations.
Membrane Manufacturer Cat. no. Absorption Detection of IgGs Detection of cytokines
Bkg Sensitivity Bkg Sensitivity
Biotrans ICN BNRQ3R Excellent qq qqq qqqqq ?
Zeta-probe Bio-Rad 162-0155 Good qq qqq
Colonyrplaque screen NEN NEF-978X Very good qq qqq
Hybond-Nq Amersham PRN303B Very good qq qqq q qqq
Magnacharge MSI NBOHY00010 Poor q qqq qqqqq q
MagnaGraph MSI NJOHY00010 Excellent y qq qqqqq q
Hybond ECL Amersham RPN2020D Excellent qq qqq y qqq
with 5% BSArTBS, membranes were incubated with fied Eagle’s medium ŽDMEM. containing 10% fetal
a single or a combination of different cytokines calf serum ŽFCS.. 184-A1N4 cells were grown at
prepared in 5% BSArTBS for 2 h at room tempera- DMEMrF12 Ž1:1. containing insulin Ž10 mgrml.,
ture. Unbounded cytokines were washed out with transferrin Ž10 mgrml., EGF Ž10 ngrml., hydrocor-
TBSr0.1% Tween 20 and TBS. Membranes were tisone Ž0.5 mgrml. and 0.5% FCS. After 48 h, the
then incubated individually or collectively with bi- complete culture medium was then replaced with
otin-conjugated anti-cytokines antibodies. Wash was DMEM containing 0.2% calf serum ŽCS.. Cells were
followed and the arrays were imaged as done in ECL
2.4. Array assay of multiple antibodies
Different species of IgGs Ž100 mgrml. were im-
mobilized onto membranes at a quantity of 0.25 ml
per spot. The arrays were then incubated with vari-
ous Donkey anti-IgGs either individually or collec-
tively, after blocking with 5% BSArTBS. Following
wash steps, membranes were incubated with rabbit
anti-Donkey IgG, which was pre-absorbed with
agarose-immobilized guinea pig IgG, goat IgG, hu-
man IgG and sheep IgG, to remove cross-reaction
components. Images were carried out with ECL.
2.5. Protein array assay for detection of cytokines
from conditioned media and sera
One milliliter of twofold diluted conditioned me-
dia and 1 ml of fivefold diluted sera were incubated
with cytokine array membranes. To prepare condi-
tioned media, human mammary epithelial cells, 184-
A1N4 and human breast cancer cells, MDA-MB-468
cells as described in our previous publication ŽHuang Fig. 1. Selection of membranes for protein arrays. Different
membranes were spotted with IgGs and other controls as indi-
et al., 1997. were plated in 100-mm tissue culture cated. The membranes were then incubated with HRP-conjugated
dishes at a density of 1 = 10 6 cells per dish. MDA- anti-bovine IgG after being blocked with 5% BSA. The signals
MB-468 cells were cultured with Dulbecco’s modi- were visualized with ECL.
4 R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13
then cultured for another 24 h. The supernatants this approach, capture proteins, either antibodies or
were collected, centrifuged at 1000 = g, aliquoted antigens, are spotted onto a membrane. The mem-
and stored at y808C until testing. Patient’s sera brane is then exposed to a sample containing protein
were obtained from Dr. Sampath Parthasarathy at of interest. The corresponding antigen binds to its
Department of Gynecology and Obstetrics, Emory cognate antibody spotted onto membrane and de-
University School of Medicine ŽSantanam et al., tected by a developing antibody.
1998.. As a first step, a simplified system was applied to
test the feasibility of this assay. Various known
specific immunoglobulins ŽIgs. were spotted onto
3. Results membrane and detected by incubation with HRP-
3.1. A simplified model: detection of HRP-con- conjugated antibodies specific to corresponding anti-
jugated antibodies gens. The signals are then visualized by ECL.
To select a suitable membrane for the assay,
To simultaneously detect multiple proteins and bovine IgG and other controls were spotted onto
antibodies, a microspot approach was developed. In several types of membranes commercially available
Fig. 2. Specificity and sensitivity of detection of HRP-conjugated anti-species-specific IgGs. ŽA. MSI magnagraph membranes immobilized
with different species-specific IgGs were incubated with individual HRP-conjugated anti-IgGs against specific species or controls as
indicated in the figure. HRP-conjugated anti-bovine IgG was spotted onto membranes as identification of orientation. ŽB. Different
concentrations of HRP-conjugated anti-bovine IgG as indicated beside each microarray row were used to test the detection sensitivity.
R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13 5
Fig. 3. Raw image data from high-density protein arrays to detect HRP-conjugated antibody. ŽA. Array layout. Membranes were spotted
with different IgGs or controls as indicated in the array layout. There are 504 spots in one array. ŽB. Membranes were incubated
individually or collectively with HRP-conjugated antibodies as indicated in ŽB..
6 R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13
ŽTable 2.. Membranes were then incubated with antibody is spotted onto membrane, serving as a
HRP-conjugated anti-bovine IgG. As shown in Fig. capture. Another corresponding antibody is labeled
1, Magnagraph and Magnacharge gave the lowest with biotin, serving as a detector. The signals are
background in the test. Magnagraph has higher ab- visualized by ECL system.
sorption capacity than Magnacharge. Therefore, Several different types of membranes as indicated
Magnagraph was selected in the assay. in Table 2 were screened for this assay. Among
The specificity of the assay was then tested. Dif- them, Hybond ECL membrane showed highest sensi-
ferent monoclonal antibodies were incubated with tivity and lowest background Ždata not shown. and
the membranes spotted with IgGs. As shown in Fig. was used in the array format analysis of cytokines.
2A, very low background levels were consistently Six cytokines were assayed using this array format.
seen in all cases. Specific recognitions were also The specificity of this assay was first demonstrated
observed in bovine IgG, chicken IgG, donkey IgG, with individual cytokine. As shown in Fig. 4, the
goat IgG, guinea pig IgG, mouse IgG, rabbit IgG and specificity was observed in all cytokine, consistent
rat IgG. Cross-reactions were noticed among goat with ELISA data. All six cytokines specifically rec-
IgG, human IgG and sheep IgG. ognized their corresponding capture antibodies and
The sensitivity of the array was demonstrated by were detected by their cognate detection antibodies.
incubation of membrane with different concentra- No cross-reaction was observed among six cytokines
tions of HRP-conjugated anti-bovine IgG. As shown and other several controls, including EGF, BSA and
in Fig. 2B, as low as 5 pgrml of HRP-conjugated buffer only. When all six cytokines were used, spe-
anti-bovine can be detected. The same sensitivity cific signals were detected in the spots immobilized
was also seen in other HRP-conjugated antibodies with six cytokines, but not in EGF, BSA and buffer
Ždata not shown.. only. Additional controls further demonstrated the
To demonstrate that this approach can be used in high specificity of this assay, including incubation of
high density array format, membranes with a total of membranes with EGF, followed by an unrelated
504 spots immobilized with different IgGs as indi- detection antibody or incubation with detection anti-
cated in Fig. 3A were incubated with single or body alone. High sensitivity of this assay was also
combination of HRP-conjugated antibodies. As achieved. The detection level is lower than 25 pgrml
shown in Fig. 3B, a total of 504 spots can be for IL-2 ŽFig. 4B.. Several other cytokines, including
simultaneously detected with similar specificity and MCP-1 and TNFa , have also shown a similar sensi-
sensitivity as lower density arrays. tivity Ždata not shown..
To detect proteins from unknown samples, condi-
3.2. Application one: simultaneous detection of mul-
tioned media collected from human mammary ep-
ithelial cells, 184-A1N4 and human breast cancer
The protein array system described above was cells, MDA-MB-468 and sera from two individual
extended to assay human cytokines. The principle of donors were assayed for their cytokine expression.
this assay is based upon the sandwich ELISA and As shown in Fig. 4C, several cytokines were differ-
ECL. A pair of antibodies, which recognize two entially expressed in different samples. As control,
different epitopes of same antigen, is used. One no signals were detected in medium alone. The
Fig. 4. Detection of cytokines in array format with high specificity and sensitivity. ŽA. Hybond ECL membranes immobilized with different
capture antibodies against different cytokines were incubated subsequently with single cytokine or all six cytokines or controls as indicated
in the figure, with corresponding biotin-conjugated anti-cytokines or controls and with HRP-conjugated streptavidin. The signals were
detected by ECL. ŽB. High sensitivity of detection is exemplified by IL-2. Membranes spotted with cytokines were incubated with different
concentrations of cytokines. The signals were then detected with biotin-conjugated anti-IL-2 and HRP-conjugated streptavidin. ŽC.
Detection of cytokine expression from conditioned media and sera. The membranes spotted with six different anti-cytokines were incubated
with twofold diluted conditioned media and fivefold diluted sera. The membranes were then incubated with a mixture of biotin-labeled
antibodies against six different cytokines. Signals were detected by ECL.
R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13 7
results clearly demonstrated the potential application The possibility of constructing high-density array
of this system in pathophysiological samples. system is demonstrated in Fig. 5. 504 spots in a
8 R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13
Fig. 5. Detection of cytokines in high-density protein array format. ŽA. Different cytokines or controls were spotted on Hybond ECL
membranes in high-density array format as indicated in the figure. ŽB. Detection of cytokines with high-density array format was
demonstrated by incubation of the array membranes with individual or collective cytokines.
R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13 9
single membrane can be simultaneously detected. All species of IgGs were spotted onto the MSI Magna-
the signals were detected as expected. graph membrane. After blocking with BSA, mem-
branes were incubated with individual donkey anti-
3.3. Application two: simultaneous detection of mul- IgGs against given species. After extensive wash to
tiple antibodies remove the unspecific binding, membranes were in-
cubated with anti-donkey IgG. Extensive wash was
The protein array system can also be used to then followed and signals were visualized with ECL.
detect antibodies. In this assay, different known anti- As shown in Fig. 6, specific signals were detected
gens are spotted onto membranes. The membranes using donkey anti-chicken, guinea pig, human,
are then incubated with samples containing antibod- mouse, rabbit and rat. Some cross-reactions were
ies to be detected. After extensive wash, the mem- observed between donkey anti-goat and sheep. The
branes are incubated with HRP-conjugated anti- sensitivity of this assay is between 5 and 50 pgrml
bodyŽies. against species-specific IgGs. The signals as demonstrated using donkey anti-mouse IgG. The
are visualized by ECL. examples of high-density arrays were demonstrated
To demonstrate that this is a feasible approach to in Fig. 7. Incubation of membranes with single or
detect multiple antibodies simultaneously, different combination of donkey anti-IgGs against specific
Fig. 6. Specific and sensitive detection of antibodies in array format. ŽA. MSI Magnagraph membranes loaded with different IgGs as
indicated were incubated with single donkey anti-species-specific IgGs or all eight antibodies. Membranes were then incubated with
HRP-conjugated anti-donkey IgG. ŽB. Different concentrations of donkey anti-mouse IgG were used to test the detection limit in array
10 R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13
Fig. 7. High-density protein arrays to simultaneously detect multiple antibodies. Different IgGs or controls were loaded onto Hybond ECL
membranes in an array format as indicated in ŽA.. Simultaneous detection of multiple antibodies was demonstrated as indicated in ŽB..
R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13 11
species produced the specific signals just as ex- fact, our unpublished data showed that the sensitivity
pected. would enhance about one order by using Amersham
plus ECL kit. Therefore, the assay system is very
sensitive. Furthermore, the intensities of signals can
4. Discussion be adjusted by exposure times, allowing detection of
high and low abundant proteins at the same time.
The array format for global analysis of gene The microspot approach makes the high-density
expression, mainly cDNA microarrays and DNA chip protein array possible. In the system described here,
technology has revolutionized biological and medical as many as 504 spots can be detected simultane-
research. Recently, the array format has been used in ously. The limitation of spots is compromised due to
systematical analysis of protein–protein interaction the manual manipulation of the immobilization pro-
ŽEmili and Cagney, 2000.. Arrays have the advan- cess. The application of a microarrayer should allow
tage of being scalable, flexible and easy to perform. for the construction of a much higher density of
The nature of arrays allows a high-throughput array system.
screening using robotic, imaging, or analytical meth- By using densitometry or CCD imaging system,
ods. However, no protein array method in the analy- one can compare the differential protein levels among
sis of protein expression has been developed. Here multiple samples. To quantitate the exact amount of
the antibody-based protein array format for analysis proteins, multiple standard curves can be generated
of multiple protein expression was described. The simultaneously. According to standard curves, the
principle of this method involves immobilization of exact amount of individual protein can be deter-
capture protein onto membrane, incubation of mem- mined.
brane with protein-containing sample and detection The concept of antibody-based microarray was
of protein of interest by exposure to a solution proposed in the mid- to late-1980s by Drs. Ekins and
containing developing antibody against a second epi- Chu ŽEkins and Chu, 1992.. They proved mathemati-
tope of protein of interest. The methodology de- cally that antibody-based arrays are able to simulta-
scribed here has several advantages over 2-D gel neously determine multiple protein levels with high
combined mass spectrometry and SELDI. It is so sensitivity ŽEkins and Chu, 1997; Ekins, 1998.. Since
simple that it can be performed at any laboratory then, several groups have attempted to establish an
setting without any sophisticated device. In contrast, antibody-based array assay to simultaneously deter-
2-D gel combined mass spectrometry requires sev- mine multiple protein expression. Among them, Dr.
eral technically difficult methodology and very ex- Silzel et al. demonstrated that multiple IgG sub-
pensive equipment. A special and expensive machine classes could be detected by fluorescence imagining
is also needed in SELDI. Secondly, the array format ŽSilzel et al., 1998.. Researchers at Genometrix fur-
allows us to detect any protein of interest. While 2-D ther demonstrated a possibility to detect multiple
gel combined mass spectrometry and SELDI is pow- IgGs in a high-throughput format ŽMendoza et al.,
erful in the determination of differential profile of 1999.. Recently, the technology of protein arrays is
protein expression, the specific protein expression is exploding ŽWalter et al., 2000; Service, 2000; Brad-
hard to detect. Finally, the antibody-based array bury, 2000.. After we completed the work described
approach is particularly useful in accurately measur- in this paper, several groups have reported to apply
ing the difference in individual protein levels be- protein array format to screen antibody–antigen in-
tween two samples, which is difficult to achieve teraction Žde Wildt et al., 2000., to study protein–
using 2-D gel approach. Finally, the background is protein interaction ŽMacBeath and Schreiber, 2000;
lower. Therefore, the sensitivity is high. The detec- Wang et al., 2000., to analyze yeast protein kinases
tion limitation for IL-2 is lower than 25 pgrml; for ŽZhu et al., 2000. and to examine autoimmune anti-
donkey anti-mouse IgG, it is between 5 and 50 bodies ŽJoos et al., 2000.. I demonstrated an alterna-
pgrml. The sensitivity is higher than ELISA. The tive and simple way to simultaneously detect multi-
sensitivity of detection will significantly increase if ple protein expression level by combination of
we apply other more sensitive ECL detection kits. In ELISA, microspot and ECL. Simplicity, low back-
12 R.-P. Huangr Journal of Immunological Methods 255 (2001) 1–13
ground and high sensitivity are among the advantage and application of protein-interacting peptides se-
over other antibody-based array approaches. The pro- lected by phage library. Nevertheless, the results
tein array system described here, which permits the presented here suggest that this is a feasible ap-
simultaneous determination of multiple proteins, will proach. With the advance of antibody engineering
prove to be of particular value in many areas. One and thousands of antibodies commercially available,
example is to study the expression of cytokines, the antibody-based protein arrays will become an
chemokines and growth factors. In this situation, the increasingly important tool in studying multiple pro-
different capture antibodies are spotted onto mem- tein expression.
brane. Membranes are then incubated with condi-
tioned media, sera or other analyte-containing fluids.
The non-specific binding is removed and the signals Acknowledgements
are revealed by a pool of developing antibodies
against different antigens. The nature of collection of This work was supported by NIHrNCI grant
samples from either conditioned media or patient CA89273 ŽRPH. and ACS grant RPG-99-164-01-
sera makes the detection of cytokines by a protein CNE ŽRPH.. We would like to express our thanks
array system much more attractive than cDNA array for the support by the Helen Dyar King Fund at the
system. Another application of protein array technol- Arizona Community Foundation for Cancer Re-
ogy described here is detection of antibodies, which search. We are grateful to Dr. Sampath Parthasarathy
is particularly useful in the study and diagnosis of for providing sera in this study.
infectious disease. In theory, this is simpler than
detection of cytokine since only one developing anti-
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