Literature Review

Protein Array Technology: Potential Use in Medical Diagnostics

Article· Literature Review (PDF Available)inAmerican Journal of PharmacoGenomics 1(1):37-43 · February 2001with 75 Reads
DOI: 10.2165/00129785-200101010-00005 · Source: PubMed
The human genome is sequenced, but only a minority of genes have been assigned a function. Whole-genome expression profiling is an important tool for functional genomic studies. Automated technology allows high-throughput gene activity monitoring by analysis of complex expression patterns, resulting in fingerprints of diseased versus normal or developmentally distinct tissues. Differential gene expression can be most efficiently monitored by DNA hybridization on arrays of oligonucleotides or cDNA clones. Starting from high-density filter membranes, cDNA microarrays have recently been devised in chip format. We have shown that the same cDNA libraries can be used for high-throughput protein expression and antibody screening on high-density filters and microarrays. These libraries connect recombinant proteins to clones identified by DNA hybridization or sequencing, hence creating a direct link between gene catalogs and functional catalogs. Microarrays can now be used to go from an individual clone to a specific gene and its protein product. Clone libraries become amenable to database integration including all steps from DNA sequencing to functional assays of gene products.
Protein Array Technology
Potential Use in Medical Diagnostics
Konrad Büssow, Zoltán Konthur, Angelika Lueking, Hans Lehrach and Gerald Walter
Max Planck Institute of Molecular Genetics, Berlin, Germany
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. From 2D Electrophoresis and Microtitre Plates to Microarrays of Biomolecules . . . . . . . . . . .
3. Protein Resources for Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Planar Immobilization of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Microfluidic Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Detection of Molecular Interactions on Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Living Protein Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Applications of Protein Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The human genome is sequenced, but only a minority of genes have been assigned a function. Whole-genome
expression profiling is an important tool for functional genomic studies. Automated technology allows high-
throughput gene activity monitoring by analysis of complex expression patterns, resulting in fingerprints of
diseased versus normal or developmentally distinct tissues. Differential gene expression can be most efficiently
monitored by DNA hybridization on arrays of oligonucleotides or cDNA clones. Starting from high-density
filter membranes, cDNA microarrays have recently been devised in chip format. We have shown that the same
cDNA libraries can be used for high-throughput protein expression and antibody screening on high-density
filters and microarrays. These libraries connect recombinant proteins to clones identified by DNA hybridization
or sequencing, hence creating a direct link between gene catalogues and functional catalogues. Microarrays can
now be used to go from an individual clone to a specific gene and its protein product. Clone libraries become
amenable to database integration including all steps from DNA sequencing to functional assays of gene products.
Am J Pharmacogenomics 2001; 1 (1)
© Adis International Limited. All rights reserved.
1. Introduction
The first complete human genome sequence is now public
knowledge. Automated technology, genomics databases and soft-
ware tools will allow the fast and efficient identification of all
estimated 100 000 human genes. The medical application of this
information is expected to lead to new generations of drugs for
the diagnostics and therapeutics markets. However, genes will
only be useful for drug development and medical diagnostics if
their functions are known. To date, genomics-based research into
common diseases has led to only a few diagnostic tools and as yet
no drugs. Progress has been hampered by the very nature of these
diseases. Common diseases, such as cancer, are generally not
caused by a single gene but by multiple genes and are further
impacted by environmental influences. The contribution of a par-
ticular gene to a disease may be relatively small, and research may
require much larger samples of individuals than would be required
in a disease caused by a single gene. In addition, some genes can
affect more than one disease. To tackle these current limitations
in the medical use of genome information, ‘functional genomics’
is now unfolding as a new research and development area. The
central idea is the annotation of the human genome with data that
can ascribe functions to genes. New technology for the analysis
of gene expression profiles of normal and diseased cells and tis-
sues, protein structures and interactions, in vitro functionality and
metabolic networks enables the first steps in this direction.
In general, the functional status of tissues of different devel-
opmental stages, differentiation and disease status correlates with
the expression of certain sets of genes. By monitoring the amounts
of transcript or protein in a cell, the expression strength of these
genes can be determined and ideally, the function of the genes
can be inferred by their level of expression. In recent years, tech-
nologies have become available which increase the throughput of
these efforts. Most prominently, gene expression patterns are
compared on the transcriptional level by DNA hybridization or
by sequencing approaches. As a consequence, expressed se-
quence tags (ESTs) for most human genes have been found and
deposited in the nucleotide databases.
However, only a minor-
ity of the proteins encoded by these sequences have yet been
assigned a function.
With the introduction of automated technologies in the field
of molecular biology and especially microarray technology, ge-
nome and gene expression analysis has been accelerated enor-
mously. Microarray technology was enabled by the development
of devices that can array biological samples at high density and
with high precision.
Oligonucleotide and cDNA microarrays
have become hot commodities for researchers, representing thou-
sands of individual genes arrayed on filter or glass slide sup-
To examine variation in gene expression, sets of oligo-
nucleotides or complex probes, generated by reverse
transcription of RNA from different tissues and cell-lines, are
hybridized on the arrays.
Multiplexed genotyping by analysis
of single nucleotide polymorphisms (SNPs) is performed with
oligonucleotide arrays by primer extension approaches. Allele-
specific arrayed oligonucleotides are extended by reverse tran-
scription using RNA from complex mixtures as the template.
cDNA microarrays have already been used to profile human tis-
sues like bone marrow, brain, prostate and heart
and complex
diseases such as rheumatoid arthritis
and cancer.
ever, DNA chip technology is still hampered by the lack of com-
mon quality standards that enable the comparison of results ob-
tained in different laboratories and with arrays of different
Nonetheless, protein chips are already emerging to
follow DNA chips as tools for automated and miniaturized func-
tional analysis.
Analogous to DNA microarrays, protein ar-
rays offer the opportunity to screen thousands of immobilized
biomolecules at a time, using steadily reduced amounts of sample
(fig. 1).
2. From 2D Electrophoresis and Microtitre Plates to
Microarrays of Biomolecules
Two-dimensional gel electrophoresis separates proteins ac-
cording to size and charge, therefore allowing the study of cell,
tissue and even whole organism proteomes.
Until recently
however, the identification of the thousands of separated proteins
RNA aptamers
Small molecules
Complex mixtures,
clinical samples
Animal immunisation
In vitro selection
Recombinant proteins
cDNA (expression) libraries
Rational design
Concentrations of biomolecules
Small molecules
Nucleic acids
Identification of novel interactions
Concentrations of biomolecules,
eg. autoimmune antibodies
Determination of antibody binding
specificity and cross-reactivity
Compex DNA
Single DNA probe,
Expression patterns on the
transcript level eg. identification
of differentially expressed genes
in tumor cells
Fig. 1. High density biomolecule arrays. Biomolecule arrays are divided into 3 categories, antibody arrays, protein/peptide arrays and DNA arrays. DNA arrays are used
to characterize cDNA libraries by DNA hybridization with single DNA probes and to determine gene expression patterns by hybridization with complex hybridization
Multiplexed genotyping is achieved by primer extension of arrayed oligos using reverse transcription with complex RNA mixtures as the transcription
The main application of arrays of antibodies, RNA aptamers or plastibodies with known binding specificities in the future will presumably be in the detection
and quantification of biomolecules as proteins, peptides or chemical compounds in complex mixtures, for example, clinical samples.
[17-22 ]
In contrast, arrays consisting of recombinant proteins or synthetic peptides are mainly used to identify and characterize interactions or biological activities of proteins
with various kinds of biomolecules, for example, by screening an arrayed expression library with an antibody of unknown binding specificity.
2 Büssow et al.
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used to be a major challenge. With the introduction of new and
automated mass spectrometric protein identification procedures,
the high throughput identification of the separated proteins is
much simplified
and allows to generate catalogues of ex-
pressed proteins in a cell or tissue of interest. Nevertheless, as the
separated proteins are obtained in denatured form and in limited
amounts, the expression of a protein of interest in recombinant
form is usually required for functional characterization. The other
classical array format in proteomics, the microtitre plate, is a well
established and still widely used standard in medical diagnostics.
To increase the number of samples and decrease reagent volume,
the 96-well microtitre plate has been developed further to plates
with 384 and 1536 wells, maintaining the original plate footprint.
As it has already occurred in DNA analysis, the microtitre plate
is now gradually being replaced by microarrays on flat surfaces
such as glass slides (‘chips’) or membranes.
The format and the preparation of protein microarrays de-
pend on the nature of the immobilized biomolecule and its appli-
cation (fig. 1). While peptide arrays are manufactured syntheti-
cally directly on the support,
proteins are delivered using
either pin-based spotting or liquid microdispensing. To date, the
most commonly arrayed proteins are antibodies, since they are
robust molecules which can be easily handled and immobilized
by standard procedures without loss of activity. Microarrays have
been developed for highly parallel enzyme-linked immunosorb-
ent assay (ELISA) applications
and could be used for parallel
analysis of multiple biomolecules within a single biological sam-
ple. For such binding studies, RNA aptamers are now arising as
an interesting alternative to antibodies. They are selected for spe-
cific binding properties in vitro (systematic evolution of ligands
by exponential enrichment, SELEX
). Using an automated
SELEX procedure, arrays of aptamers are being developed and
can complement antibody arrays to specifically detect
biomolecules in complex mixtures.
Another future binding
format are specific recognition sites in polymers created by mo-
lecular imprinting using template molecules.
The goal of
this approach is to obtain surfaces with moulds, so called ‘plas-
tibodies’, which fit only certain protein shapes and therefore bind
these shapes specifically. Surfaces specifically binding BSA,
IgG, ribonuclease A or lysozyme from two-component mixtures
have been described.
3. Protein Resources for Arrays
For protein arrays, resources of large numbers of proteins,
preferably in purified form, represent a major technical challenge.
A highly parallel and automated approach to protein expression
is required. While in prokaryotes genomic fragments can be di-
rectly cloned into expression vectors, this is not possible in eu-
karyotes due to the presence of introns in their genes. The recom-
binant expression of all open reading frames of the yeast Saccha-
romyces cerevisiae has recently been achieved,
and a nearly
complete collection of yeast strains for the expression of 6144
open reading frames as fusion proteins was generated, divided in
pools and screened for biological activities. Collections like this
can form the basis of future protein microarrays by representing
a large proportion of gene products. Using a similar expression
strategy, Zhu et al.
have created protein arrays of S. cerevisiae
kinases. In total, 119 protein kinases were expressed, purified as
glutathione S-transferase (GST) fusion proteins, arrayed and
cross-linked in a protein chip format and assayed for au-
tophosphorylation by treatment with radiolabeled ATP. Substrate
specificity was assayed with protein chips each carrying one of a
set of kinase substrates. The kinases and the radiolabeled ATP
were arrayed by pipetting onto the substrate coated surfaces and
phosphorylation was monitored.
In less well-known systems such as human tissues, full-
length cDNA clones must be isolated before protein expression
can be started. High-throughput subcloning of open reading
frames has been described
but remains a major difficulty if
complete proteomes of higher organisms are to be studied. To
overcome these problems, arrayed cDNA expression libraries,
cloned in bacterial and yeast expression vectors, have been de-
veloped in our laboratory. These libraries are characterized by
standard DNA hybridization and sequencing techniques,
screened for properties of their expression products and hence,
represent a source for large numbers of recombinant pro-
In addition, expression libraries eliminate the need
to construct individual expression systems for every protein of
interest. By arraying, the expression products of complete librar-
ies can be characterized in parallel. On the other hand, a large
proportion of clones do not express their insert in a suitable form,
mainly due to cDNA fragments being fused to the vector-encoded
start codon in the wrong reading frame. Therefore, non-expres-
sion clones have to be identified and removed from the library.
To identify expression clones, hundreds of thousands of clones
are arrayed on filter membranes and protein expression is in-
duced. By detection of a His
-tag peptide fused to the protein
products, desired expression clones are identified and rearrayed
into a new library.
Our protein filter array technology was further developed to
increase spot density and to facilitate the arraying of purified
proteins. Lueking et al.
have used automated arraying from
liquid expression cultures using either a new pin-based or a high-
speed picoliter dispensing (inkjetting) device mounted onto a
flat-bed gridding robot. For this purpose, 96 proteins of the hu-
Protein Array Technology 3
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man foetal brain cDNA library hEx1
were expressed in liquid
bacterial cultures, and solutions were spotted onto polyvinylidene
difluoride (PVDF) filters, either as crude lysates or after purifi-
cation by Ni-NTA immobilized metal affinity chromatography
(IMAC). 4800 samples were placed onto polyacrylamide-coated
microscopic slides and simultaneously screened, using a hybrid-
ization automat, applying minimal amounts of reagents (less than
100 µl antibody solution; Lueking, [unpublished observations]).
Sharp and well-localized signals allowed the detection of 250
attomol or 10 pg of a spotted test protein (GAPDH, glyceralde-
hyde-3-phosphate dehydrogenase, Swiss-Prot P04406).
To achieve standardized microarrays carrying thousands of
verified recombinant proteins, high-throughput methods for pro-
tein expression and purification are required, as well as a pipeline
for the identification and verification of expression products. By
combining protein expression and purification in array
(microtitre plate) format with high-throughput protein mass de-
termination by mass-spectrometry, large numbers of library
clones have been identified and their expression products charac-
This approach can be used to both identify unknown
clones from expression libraries and to verify expression products
generated at high-throughput.
4. Planar Immobilization of Proteins
Proteins are delivered onto solid supports by either pin-based
spotting or microdispensing devices. The technique of immobi-
lization is important both for effective concentration and orienta-
tion of immobilized proteins or antibodies on the surface. A va-
riety of methods have been reported, including the adsorption to
charged or hydrophobic surfaces, covalent cross-linking or spe-
cific binding via tags (e.g. nickel chelating or streptavidin coated
surfaces for Plasmon Resonance measurements, Biacore AB, Up-
psala, Sweden).
Flat pins are routinely used for spotting of nanoliter volumes
of proteins, as flat pins are less sensitive to variation of sample
viscosity than slit pins or microdispensing systems.
A slit pin
arraying device was used by MacBeath and Schreiber
to pro-
duce a microarray of 10,800 spots of 2 distinct proteins (protein
G and an FKBP12 binding domain), which were then specifically
detected with fluorescently labeled IgG and FKBP12, respec-
tively. As an alternative to metal pins, Martin et al.
reported a
hydrogel ‘stamper’ for disposition of sub-monolayers of antibod-
ies. They were immobilized on an aminosilyated surface and re-
tained their binding activity. In addition, other approaches to im-
mobilise proteins like BSA, avidin or monoclonal antibodies
have been reported using either photolithography of silane mono-
or gold,
combining microwells with microsphere
or inkjetting onto polystyrene film.
Protein immo-
bilization on flat surfaces was achieved by either covalent cou-
pling to a crosslinker attached to the surface,
or noncova-
lent interaction to an immobilized biomolecule.
The biotin/avidin system is the most frequently used nonco-
valent immobilization system, due to the extraordinary high af-
finity of the biotin-avidin interaction. Since avidin (or
streptavidin) homotetramers bind up to 4 biotin molecules, they
can be used to link biotinylated macromolecules to a surface that
was chemically coated with biotin.
Proteins are either
biotinylated in vitro using commonly available reagents, or in
vivo by expression of proteins fused to a peptide biotinylation
The density of protein molecules immobilized on the support
is mainly determined by the surface structure. A flat, 2-dimen-
sional surface offers less binding capacity than the 3-dimensional
structure of a filter membrane or a polyacrylamide gel layer.
Mirzabekov and co-workers produced 3-dimensional polyacryl-
amide gel pad microarrays providing an immobilization capacity
more than 100 times greater than that of 2-dimensional glass sup-
ports, thus increasing the sensitivity of measurements consider-
The gel pads are separated by a hydrophobic glass sur-
face and provide a native, aqueous environment and can
accommodate proteins of up to 400 kiloDalton in size.
matic activity of several enzymes like horseradish peroxidase,
alkaline phosphatase and β-D-glucuronidase has been detected in
these hydrogel pads. Prestructured surfaces consisting of hydro-
philic spots on hydrophobic surfaces have also been reported for
protein arraying.
The hydrophobic surface prevents the aque-
ous drops applied to the hydrophilic spots from mixing.
An array based on the 96-well microtitre plate footprint was
developed by Mendoza at al.,
consisting of 96 6x6 microarrays
printed with a pressure-controlled multi-capillary device. In com-
bination with a custom scanning charge-coupled device (CCD)
detector, the microarrays are used for multiplexed ELISAs. 20
samples plus controls are subjected in parallel to 96 different
ELISAs by this system.
Recently, a high-precision sub-microliter liquid dispensing
system has been developed for the preparation of hanging drop
arrays for protein crystallization. These arrays consist of 2µl to
100nl drops and are used to screen for suitable buffer and salt
conditions for protein crystallization.
5. Microfluidic Devices
Planar microarrays are a robust and easy-to-handle format
for the miniaturized immobilization of large numbers of analytes.
Nonetheless, the introduction of 3-dimensional microstructures
4 Büssow et al.
Adis International Limited. All rights reserved. Am J Pharmacogenomics 2001; 1 (1)
on a chip offers a number of additional options for experimenta-
tion (for a recent review see Sanders & Manz
). Such
microfluidic devices are equipped with channels for transporting
reagents to immobilized target molecules. For example, an assay
for Protein Kinase A was developed on a microfluidic chip,
where all necessary reagents were placed in cavities on the chip
and delivered to the reaction chamber through micro-channels.
Microfluidic chips do certainly offer specific advantages over
planar microarrays but due to their complex production proce-
dures, their development and applications are still at an early
6. Detection of Molecular Interactions on
On DNA microarrays, hybridization events are detected us-
ing fluorescently or radioactively labeled probe molecules.
corresponding approach for the detection of protein-protein, pro-
tein-DNA and protein-small molecule interactions has been re-
ported. The ‘universal protein array system’ (UPA), consists of
filter membrane arrays of purified proteins.
Specific binding
properties of the immobilized proteins on the low-density UPA
arrays were demonstrated with various radiolabeled protein,
DNA, RNA and small molecule ligands. By washing the mem-
brane under different salt conditions, high-affinity protein-pro-
tein interactions could be distinguished.
In addition to fluorescent dyes and radioisotopes, a wide
range of detection options exists for protein and antibody arrays
(reviewed in Rogers
). Unlabeled ligands can be identified in-
directly by using a secondary antibody (sandwich assay). As an
alternative to these noncompetitive formats, various competitive
assays, relying on competition of the ligand with labeled tracers,
are in use. Tracers are either detected directly on the chip or
unbound tracer molecules are measured in solution. Protein chips
for direct measurement of protein mass by matrix-assisted laser
desorption-ionization time-of-flight (MALDI-TOF) mass spec-
trometry have been described.
Also reported were surface
enhanced laser desorption-ionization (SELDI, Ciphergen
Biosystems) protein chips coated with either antibodies
charged or hydrophobic groups
for protein adsorption, fol-
lowed by MALDI-TOF mass-spectrometry on the chip to directly
identify the captured polypeptides. Another method for the direct
detection of unlabeled ligand binding employs surface plasmon
resonance (SPR).
Using online detection in flow cells, this
technology allows the determination of binding rates and disso-
ciation constants. In addition to antibodies, SPR detection of pro-
tein-protein interaction on microarrays has been described.
7. Living Protein Arrays
In contrast to oligonucleotide or peptide arrays, proteins can-
not be synthesized directly on the support, but usually are gridded
out of microtitre plates. Alternatively, cell arrays have been re-
ported in which proteins of interest are produced by induction in
bacterial or yeast expression hosts.
Recently, a comprehensive
analysis of protein-protein interactions in S. cerevisiae was un-
dertaken by yeast 2-hybrid screens in array format.
So called
‘living arrays’ were constructed consisting of a nearly complete
set of yeast open reading frames cloned as fusions with the Gal4
activation domain. This clone set was co-transformed with a set
of putative interaction partners cloned as fusions to the Gal4 DNA
binding domain and subsequently arrayed on filter membranes.
Protein-protein interaction was detected by arraying of the co-
transformed clone set on selective media. By screening 5345
yeast open reading frame-Gal4 activation domain fusions with
195 Gal4 DNA binding domain fusions, 957 putative interac-
tions, involving 1004 yeast proteins, were identified.
8. Applications of Protein Arrays
A large variety of assays have been adapted to utilize protein
microarrays. Currently however, the detection of immobilized
antigens with antibodies is still the most common application (fig.
1). Protein and antibody arrays have been used for the selection
and characterization of novel antibodies from phage display li-
braries and for the identification of antigens (e.g. involved in
autoimmune diseases).
Phage display antibody libraries have been developed for the
in vitro selection of antibodies as an alternative to animal immu-
nization (reviewed in Holt et al.
and Hoogenboom et al.
For this purpose, recombinant immunoglobulin gene libraries are
cloned in phagemid vectors and antibody fragments are displayed
as fusion proteins on the surface of bacteriophage (reviewed in
). Recently, protein arrays of our cDNA expression li-
brary hEx1
were used to identify antigens recognized by ran-
domly selected antibody fragments from a phage display anti-
body library.
By screening 12 different antibody fragments on
an array of 27 000 expression clones, 4 novel and highly specific
antigen-antibody pairs were detected. In a related approach, an-
tibody arrays were used for the identification of specific anti-
body-producing bacteria.
For this purpose, bacteria containing
phagemid selected from a phage antibody library by in vitro pan-
ning on chosen antigens were arrayed on filter membranes. After
cell growth, antibody production was induced and specifically
binding antibodies were captured and identified on a second, an-
tigen coated membrane. By screening 18 342 antibody clones at
Protein Array Technology 5
Adis International Limited. All rights reserved. Am J Pharmacogenomics 2001; 1 (1)
a time, highly specific antibodies were selected after just one
round of panning.
In autoimmune disorders, self-reacting antibodies (i.e. pro-
duced against the organism’s own proteins and epitopes) play an
important role in the clinical manifestation of the diseases. There-
fore, profiling the antibody repertoire of patients with autoim-
mune disease is believed to be medically relevant and informa-
tive. Characterization of autoimmune patient sera on protein
chips would allow the diagnosis of autoimmune diseases based
upon the presence of specific autoantibodies. For the identifica-
tion of antigens recognized by autoantibodies, sera from patients
with autoimmune disease have been hybridized to un-
characterized λgt11 cDNA phage libraries or to tissue extracts
separated by 1D or 2D gel electrophoresis.
The subsequent
characterization of the identified antigens is labor intensive, also
requiring expensive sequencing of the identified proteins. Such
characterization resulted in attribution of novel functions to these
proteins and in some cases, suggested their potential as therapeu-
tic targets.
To simplify the characterization of autoantibodies,
serum can be applied to protein arrays containing large numbers
of recombinant proteins of known identity. Moreover, using pro-
tein arrays will overcome the problems associated with protein
level variation in natural tissue extracts and hence increase repro-
ducibility. The use of protein chips allows for the determination
of the binding profile of autoimmune antibodies of each patient
and for each disease. Once disease-specific antigens are known,
it is possible to create a diagnostic protein array. Recently, Joos
et al.
reported a micro-array based test for the parallel detec-
tion of autoantobodies in human sera.
In contrast to the stand-
ard test involving time consuming tissue or cell culture immuno-
fluorescence, only minimal amounts of sera (1µl per sample) are
required. 35 clinically characterized patients were assayed on
protein arrays with 20 different antigens and several control pro-
teins spotted in various dilutions to confirm and analyze their
As shown by Lueking et al.,
apparently specific monoclo-
nal antibodies (α-HSP90, α-β-tubulin) showed considerable
cross-reactivity with other proteins following incubation on pro-
tein microarrays, consisting of 96 in liquid bacterial cultures ex-
pressed proteins of the hEx1 library. In a way, this is not surpriz-
ing, as antibodies are not usually tested against whole libraries of
proteins. However, in immunohistochemical or physiological
studies against whole cells or tissue extracts, this cross-reactivity
of antibodies can lead to false interpretations. Therefore, the char-
acterization of the binding specificity of antibodies used exten-
sively in diagnostic tools, is of prime importance.
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Protein Array Technology 7
Adis International Limited. All rights reserved. Am J Pharmacogenomics 2001; 1 (1)
  • ... Protein analysis often comes with its own issues including, but not limited to, consideration of temporal and spatial changes as well as the physiological state of the protein set when samples were collected. The field has grown from 2D gel electrophoresis (2DE) and microtiter plates to mass spectrometry (MS) and microarrays [55], dramatically increasing throughput and proteome coverage over time. ...
    While biomarker discovery is receiving increased attention in both drug and chemical safety assessment, a critical step is transitioning a biomarker from an experimental observation to a useful assay is that of analytical validation. This is particularly relevant as one moves from biomarker discovery using an ‘omics platform to exploratory use as implemented in a more focused platform. Large scale discovery platforms for transriptomics, such as microarrays, have unique analytical considerations, different from that of more focused platforms, such as qRT-PCR. Likewise, mass spectrometry based screens of the proteome have different analytical challenges as compared to focused ELISA assays. Metabolomics experiments also require attention to analytical validation, with different considerations for quantitative or semi-quantitative assays. And while guidelines for analytical validation are still evolving, general principles certainly apply, with a solid body of literature that should be consulted by the biomarker scientist before deeming an assay as worthy of more widespread use.
  • ... Important choices also need to be made regarding the surface chemistries on to which the arrays are fabricated, in order to ensure that sufficient protein densities per spot are achieved, as well as to control orientation and folding of arrayed target proteins when required. The simplest fabrication approaches make use of either chemically-reactive glass surfaces (typically either aldehyde-, N-hydroxy succinamide-or epoxide-activated) or of nitrocellulose-coated glass [8,19]; in both cases, immobilization of essentially all proteins can be readily achieved in parallel, but the binding to such surfaces is uncontrolled, through random covalent coupling or non-specific physisorption, respectively and can lead to unfolding and loss of activity. Other approaches therefore aim to achieve controlled, oriented immobilization of different recombinant proteins onto the surface via a common affinity tag [2,12,14,17,20,21], which has theoretical advantages, particularly if downstream, quantitative, on-array functional assays are required [20]. ...
    Introduction: High-content protein microarrays in principle enable the functional interrogation of the human proteome in a broad range of applications, including biomarker discovery, profiling of immune responses, identification of enzyme substrates, and quantifying protein-small molecule, protein-protein and protein-DNA/RNA interactions. As with other microarrays, the underlying proteomic platforms are under active technological development and a range of different protein microarrays are now commercially available. However, deciphering the differences between these platforms to identify the most suitable protein microarray for the specific research question is not always straightforward. Areas covered: This review provides an overview of the technological basis, applications and limitations of some of the most commonly used full-length, recombinant protein and protein fragment microarray platforms, including ProtoArray Human Protein Microarrays, HuProt Human Proteome Microarrays, Human Protein Atlas Protein Fragment Arrays, Nucleic Acid Programmable Arrays and Immunome Protein Arrays. Expert commentary: The choice of appropriate protein microarray platform depends on the specific biological application in hand, with both more focused, lower density and higher density arrays having distinct advantages. Full-length protein arrays offer advantages in biomarker discovery profiling applications, although care is required in ensuring that the protein production and array fabrication methodology is compatible with the required downstream functionality.
  • ... By contrast, the biotinstreptavidin methodology used by our group is a non-covalent immobilisation method based on the high af fi nity of the biotin and streptavidin interaction. This method links biotinylated macromolecules to a surface that was previously derivatised with streptavidin via a single point of attachment ( Fig. 3.1 ) ( MacBeath and Schreiber 2000 ;Büssow et al. 2001 ) (see Supplementary Material for detailed protocol: Methodology, Sect. 1.4 ). ...
    Protein microarrays have many potential applications in the systematic, quantitative analysis of protein function, including in biomarker discovery applica-tions. In this chapter, we review available methodologies relevant to this fi eld and describe a simple approach to the design and fabrication of cancer-antigen arrays suitable for cancer biomarker discovery through serological analysis of cancer patients. We consider general issues that arise in antigen content generation, microar-ray fabrication and microarray-based assays and provide practical examples of experimental approaches that address these. We then focus on general issues that arise in raw data extraction, raw data preprocessing and analysis of the resultant preprocessed data to determine its biological signi fi cance, and we describe compu-tational approaches to address these that enable quantitative assessment of serologi-cal protein microarray data. We exemplify this overall approach by reference to the creation of a multiplexed cancer-antigen microarray that contains 100 unique, puri fi ed, immobilised antigens in a spatially de fi ned array, and we describe speci fi c methods for serological assay and data analysis on such microarrays, including test cases with data originated from a malignant melanoma cohort.
  • Chapter
    Polymeric-patterned surfaces are finding significant importance in various biomedical applications such as screening and diagnostic assays, tissue engineering, biosensors, and in the study of fundamental cell biology. A wide variety of methods, involving photolithography, inkjet printing, soft lithography, and dip-pen lithography, have emerged for protein or polymer patterning on various substrates. For directional immobilization or adsorption of protein, surface requires pre-defined regions to which protein molecules can be immobilized. The most common techniques to introduce defined protein immobilization are soft lithography and photolithography. However, these techniques have some associated limitations. In soft lithography, stamps with well-defined structures are required, and the migration of ink during and after printing needs to be well controlled. In photolithography, a polymeric photoresist and a mask are needed which require expensive setup to fabricate. Therefore, facile and economic techniques are worth exploring. The dewetting of a thin polymeric film is a spontaneous and self-organized process that forms an array of microscale and nanoscale droplets on a substrate. This is a facile approach of patterning polymer on glass substrate providing a reliable surface for specific, dense, and uniform immobilization of desired molecules to pre-designed patterns. Since antibody orientation is very important in antibody-based surface capture assays, patterned polymer surfaces are of great importance with respect to an increasing number of biosensor applications. Apart from protein patterning, such polymeric-patterned surface can be effectively used in specific type of cell isolation and detection. Indeed, it is found that circulating tumor cells (CTCs) are easily isolated using such patterned structures either on a flat plate or inside a microfluidic environment.
  • Chapter
    Further methodological developments particularly in the field of the array technology enable new approaches for the identification and characterization of tumour relevant proteins. We used a proteomic-based approach to search for tumour specific target molecules in pancreatic carcinoma tissue and present data on some promising candidates with respect to their tumour biological value. The expression of tumour-associated antigens can provoke an autologous immune response in patients suffering from cancer and thus enables a systematic screening of patient sera to reveal promising proteins. Sera from pancreatic carcinoma patients were incubated with the high-density protein array Library 800 obtained from the Resource Center of the Human Genome Project (RZPD; This protein array consists of gridded bacteria clones expressing more than 37 000 different peptide sequences and proteins, which are representing around 10 000 to 12 000 different genes. Overall, 65 bacterial clones were detected positive after incubation with serum (n = 10) from patients with advanced pancreatic carcinoma. The DNA sequencing results showed a collection of uncharacterized, hypothetical or well characterized proteins. The well characterized proteins were for example Pur-1 (myc-associated zinc finger protein), the melanoma-associated antigen F1 (MAGE-F1) and alpha-tubulin. The protein filter approach is a suitable tool for the serological identification of tumour antigens which was confirmed by candidate matches obtained with autologous serological screening methods published for other cancer types in the SEREX database of the Ludwig Institute for Cancer Research (
  • Chapter
    Biomolecular patterning at the nanoscale is currently a very active field that combines the know-how of two different fields: biology and nanotechnology. The interest in the preparation of nanoscale features at surfaces relies on their wide range of applications in different areas, including physical science and life science. In particular, the immobilization of biomolecules having nanometer resolution has been pursued for their interest in fundamental studies in molecular and cell biology or to prepare platforms with different biomedical applications, such as molecular diagnostics just to mention a few of them.
  • Article
    The bio-arrays are a versatile technology with wide possibilities for the scientific advance in the XXI century. Its application had shown a significant growth in the last years and, together with this, the genetic analysis on the scientific research. In the same way that the microchip had impressed velocity to the development of the computerized science, the bio-arrays give velocity to the molecular biology and the science in general. These represent thousands of samples contained on a support in an organized way and, due to the tendency to be use it as a miniaturized form, will be possible interrogate the genome in only one bio-arrays experiment. The hybridization of these supports of bio-array allows monitor the expression and the sequence of the genes in them organized, being processed and interpreted its results through software's designed for these ends. Most of the current applications of this technology are related with the biomedical science, but their future use is of unimaginable dimensions for the current science.
  • Article
    Based on the principle of immobilized metal affinity chromatography (IMAC), it has been found that a Ni-Co alloy-coated protein chip is able to immobilize functional proteins with a His-tag attached. In this study, an intelligent computational approach was developed to promote the performance and repeatability of a Ni-Co alloy-coated protein chip. This approach was launched out of L18 experiments. Based on the experimental data, the fabrication process model of a Ni-Co protein chip was established by using an artificial neural network, and then an optimal fabrication condition was obtained using the Taguchi genetic algorithm. The result was validated experimentally and compared with a nitrocellulose chip. Consequentially, experimental outcomes revealed that the Ni-Co alloy-coated chip, fabricated using the proposed approach, had the best performance and repeatability compared with the Ni-Co chips of an L18 orthogonal array design and the nitrocellulose chip. Moreover, the low fluorescent background of the chip surface gives a more precise fluorescent detection. Based on a small quantity of experiments, this proposed intelligent computation approach can significantly reduce the experimental cost and improve the product's quality. © 2015 Society for Laboratory Automation and Screening.
  • Chapter
    The article contains sections titled:
Literature Review
  • Article
    Currently there are over 1000000 human expressed sequence tag (EST) sequences available on the public database, representing perhaps 50-90% of all human genes. The cDNA microarray technique is a recently developed tool that exploits this wealth of information for the analysis of gene expression. In this method, DNA probes representing cDNA clones are arrayed onto a glass slide and interrogated with fluorescently labeled cDNA targets. The power of the technology is the ability to perform a genome-wide expression profile of thousands of genes in one experiment. In our review we describe the principles of the microarray technology as applied to cancer research, summarize the literature on its use so far, and speculate on the future application of this powerful technique.
  • Article
    The overall history and recent advances in surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology is reviewed herein. Fundamentals of SELDI-TOF analysis are presented while drawing comparisons with other laser-based mass spectrometry techniques. The application of SELDI-TOF-MS to functional genomics and biomarker discovery is discussed and exemplified by elucidating a biomarker candidate for prostatic carcinoma. Finally, a short discussion regarding future SELDI requirements and developments is supplied.
  • Article
    This study describes a practical system that allows high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on primer arrays. The method relies on the sequence-specific extension of two immobilized allele-specific primers that differ at their 3′-nucleotide defining the alleles, by a reverse transcriptase (RT) enzyme at optimized reaction conditions. We show the potential of this simple one-step procedure performed on spotted primer arrays of low redundancy by generating over 8000 genotypes for 40 mutations or SNPs. The genotypes formed three easily identifiable clusters and all known genotypes were assigned correctly. Higher degrees of multiplexing will be possible with this system as the power of discrimination between genotypes remained unaltered in the presence of over 100 amplicons in a single reaction. The enzyme-assisted reaction provides highly specific allele distinction, evidenced by its ability to detect minority sequence variants present in 5% of a sample at multiple sites. The assay format based on miniaturized reaction chambers at standard 384-well spacing on microscope slides carrying arrays with two primers per SNP for 80 samples results in low consumption of reagents and makes parallel analysis of a large number of samples convenient. In the assay one or two fluorescent nucleotide analogs are used as labels, and thus the genotyping results can be interpreted with presently available array scanners and software. The general accessibility, simple set-up, and the robust procedure of the array-based genotyping system described here will offer an easy way to increase the throughput of SNP typing in any molecular biology laboratory.
  • Article
    We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available ( and can be used by other researchers as a digital expression reference. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AL360374–AL36537.]
  • Article
    Simultaneous parallel syntheses at distinct positions on a membrane support is exemplified with the preparation of series of predefined, short peptide sequences (1). Cellulose paper sheets are used as absorptive membranes. Peptides are assembled by manual or automated spotting of small aliquots of solutions containing the activated amino acid derivatives onto marked positions on the sheets. The application of this method to rapid epitope analysis is demonstrated.
  • Article
    Full-text available
    cDNA microarray technology is used to profile complex diseases and discover novel disease-related genes. In inflammatory disease such as rheumatoid arthritis, expression patterns of diverse cell types contribute to the pathology. We have monitored gene expression in this disease state with a microarray of selected human genes of probable significance in inflammation as well as with genes expressed in peripheral human blood cells. Messenger RNA from cultured macrophages, chondrocyte cell lines, primary chondrocytes, and synoviocytes provided expression profiles for the selected cytokines, chemokines, DNA binding proteins, and matrix-degrading metalloproteinases. Comparisons between tissue samples of rheumatoid arthritis and inflammatory bowel disease verified the involvement of many genes and revealed novel participation of the cytokine interleukin 3, chemokine Groα and the metalloproteinase matrix metallo-elastase in both diseases. From the peripheral blood library, tissue inhibitor of metalloproteinase 1, ferritin light chain, and manganese superoxide dismutase genes were identified as expressed differentially in rheumatoid arthritis compared with inflammatory bowel disease. These results successfully demonstrate the use of the cDNA microarray system as a general approach for dissecting human diseases.
  • Article
    We describe a method to pattern proteins onto a photolabile “caged” biotin-derivatized self-assembled monolayer (SAM) on gold, which we term light-activated affinity micropatterning of proteins (LAMP). LAMP is a multistep patterning process with considerable flexibility in its implementation. First, a reactive SAM on gold is formed from a mixture of 11-mercaptoundecanol and 16-mercaptohexadecanoic acid. Next, the carboxylic acid end groups in the SAM are coupled to methyl α-nitropiperonyloxycarbonyl biotin succinimidyl ester (caged biotin ester) through a diamine linker. The caged biotin is then deprotected in regions irradiated by masked UV light, and subsequent incubation with streptavidin results in selective binding of streptavidin to the irradiated regions. Micropatterning of various proteins has been demonstrated with a spatial resolution of 6 μm by confocal microscopic imaging of fluorophore-labeled proteins, and a contrast ratio of 4:1 was determined by direct ellipsometric imaging of streptavidin. Immobilization of biotinylated antibodies on the streptavidin pattern indicates that LAMP can enable spatially resolved micropatterning of different biomolecules by repeated cycles of spatially defined photodeprotection of biotin, streptavidin incubation, followed by immobilization of the biotinylated moiety of interest.
  • Article
    Micropatterned arrays of active proteins are vital to the next generation of high-throughput multiplexed biosensors and advanced medical diagnostics. We have developed a simple method for fabricating antibody arrays using a micromolded hydrogel “stamper” and an aminosilylated receiving surface. The stamping procedure permits direct protein deposition and micropatterning while avoiding cross-contamination of separate patterned regions. Three different antibodies were stamped in adjacent arrays of 50−80 μm circular areas with retention of activity. 125I labeling and atomic force microscopy studies showed that the stamper deposited protein as a submonolayer. The fluorescent signal-to-background ratio of labeled bound antigen was greater than 25:1.
  • Article
    Full-text available
    Currently there are over 1000000 human expressed sequence tag (EST) sequences available on the public database, representing perhaps 50-90% of all human genes. The cDNA microarray technique is a recently developed tool that exploits this wealth of information for the analysis of gene expression. In this method, DNA probes representing cDNA clones are arrayed onto a glass slide and interrogated with fluorescently labeled cDNA targets. The power of the technology is the ability to perform a genome-wide expression profile of thousands of genes in one experiment. In our review we describe the principles of the microarray technology as applied to cancer research, summarize the literature on its use so far, and speculate on the future application of this powerful technique.