Protein arrays are rapidly becoming established as a powerful
means to detect proteins, monitor their expression levels,
and investigate protein interactions and functions. They are
seeing an explosive progress and interest at the moment and
have become one of the most active areas emerging in biotechnology
today. The objective behind protein array development is to
achieve efficient and sensitive high throughput protein analysis,
carrying out large numbers of determinations in parallel by
automated means. While they were conceived originally as miniaturised
immunoassays, further development is being driven by genome
projects on the one hand and improved expertise in expression
of recombinant proteins on the other. Protein arrays make
possible the parallel multiplex screening of thousands of
interactions, encompassing protein-antibody, protein-protein,
protein-ligand or protein-drug, enzyme-substrate screening
and multianalyte diagnostic assays. In the microarray or chip
format, such determinations can be carried out with minimum
use of materials while generating large amounts of data. Moreover,
since most proteins are made by recombinant methods, there
is direct connectivity between results on protein arrays and
DNA sequence information.
At
the present time, protein arrays are poised to become a central
proteomics technology, important both in basic research and
commercially for biotechnology enterprises. It is well recognised
that the complexity of the human proteome far exceeds that
of the genome. When variables such as alternative gene splicing
events and post-translational modifications are taken into
account, the number of different molecular protein species
in man is likely to be at least an order of magnitude greater
than the number of genes, i.e. about 500,000 proteins. Proteomics
investigations are at the leading edge of functional genomics
today and the development of protein arrays reflects the realisation
that functional genomics discoveries will depend heavily on
progress in defining the expression of, and interactions among,
proteins. Conventional proteome analysis by 2D gel electrophoresis
and mass spectrometry, while highly effective, has limitations
and in particular may miss many proteins of interest when
expressed at low abundance. There is therefore an acknowledged
need for other sensitive and more accessible high throughput
technologies for protein detection, quantitation and differential
expression analysis in health and disease. For this reason,
protein arrays are generating enormous interest at the research
and biotechnology levels.
Microarray
ELISA-style assays will accelerate immunodiagnostics significantly.
This aspect of the technology was discussed in the 1980s by
Ekins, who introduced the concept of the ambient analyte assay
and demonstrated that microspot immunoassays could be perfomed
with high sensitivity and selectivity. In addition to diagnostics
applications, protein array technology promises to accelerate
basic research on protein-protein interactions and will allow
protein expression profiling, ranging from limited numbers
of proteins up to global proteomic analysis, while in the
pharmaceutical industry protein arrays can be integrated into
target identification and validation processes. However, as
with other high throughput functional genomics technologies,
there are major technical demands which will need to be solved
in order to achieve maximum capability. This page will attempt
to keep abreast of developments in protein array technologies,
chemistries and formats, and to provide an up to date guide
to the relevant basic research,
literature,meetings and biotech
companies.
(Note, in the text below, mention is made of specific companies
developing protein arrays; links to their websites can be
found on the companies
page. While specific literature references are not annotated
in the text, a comprehensive literature
list is provided.)
Basic
research
Defining
characteristics
Protein arrays are solid-phase ligand binding assay systems
using immobilised proteins on surfaces which include glass,
membranes, microtiter wells, mass spectrometer plates, and
beads or other particles. The assays are highly parallel (multiplexed)
and often miniaturised (microarrays, protein chips). Their
advantages include being rapid and automatable, capable of
high sensitivity, economical on reagents, and giving an abundance
of data for a single experiment. Bioinformatics support is
important; the data handling demands sophisticated software
and data comparison analysis. Fortunately some of the software
can be adapted from that used for DNA arrays, as can much
of the hardware and detection systems.
Areas
of application One of the chief formats is thecapture array,
in which ligand-binding reagents, which are usually antibodies
but may also be alternative protein scaffolds, peptides or
nucleic acid aptamers, are used to detect target molecules
in mixtures such as plasma or tissue extracts. In diagnostics,
capture arrays can be used to carry out multiple immunoassays
in parallel, both testing for several analytes in individual
sera for example and testing many serum samples simultaneously.
In proteomics, capture arrays will be used to quantitate and
compare the levels of proteins in different samples in health
and disease, i.e. protein expression profiling, and in this
the protein arrays may challenge 2DE technology. Proteins
other than specific ligand binders are used in the array format
for in vitro functional interaction screens such as
protein-protein, protein-DNA, protein-drug, receptor-ligand,
enzyme-substrate, etc. They may also be used to correlate
the polymorphic changes resulting from SNPs with protein function.
The capture reagents themselves will need to be selected and
screened against many proteins, which can also be done in
a multiplex array format against multiple protein targets.
Thus,
broadly speaking, there are at least four major areas where
protein arrays are being applied, each of which requires appropriate
formats and readout methods.
Diagnostics:
detection of antigens and antibodies in blood samples; profiling
of sera to discover new disease markers; environment and
food monitoring.
Proteomics:
protein expression profiling; organ and disease specific
arrays.
Isolation
of individual members from display libraries for further
expression or manipulation: selection of antibodies and
protein scaffolds from phage or ribosome display libraries
for use in capture arrays.
Protein
functional analysis: protein-protein interactions; ligand-binding
properties of receptors; enzyme activities; antibody cross
reactivity and specificity, epitope mapping.
Protein
sources
For construction of arrays, sources of proteins include cell-based
expression systems for recombinant proteins, purification
from natural sources, production in vitro by cell-free
translation systems, and synthetic methods for peptides. Many
of these methods can be automated for high throughput production.
For capture arrays and protein function analysis, it is important
that proteins should be correctly folded and functional; this
is not always the case, e.g. where recombinant proteins are
extracted from bacteria under denaturing conditions. Nevertheless,
arrays of denatured proteins are useful in screening antibodies
for cross-reactivity, identifying autoantibodies and selecting
ligand binding proteins.
Formats
and surfaces
Protein arrays have been designed as a miniaturisation of
familiar immunoassay methods such as ELISA and dot blotting,
often utilising fluorescent readout, and facilitated by robotics
and high throughput detection systems to enable multiple assays
to be carried out in parallel. Commonly used physical supports
include glass slides, silicon, microwells, nitrocellulose
or PVDF membranes, and magnetic and other microbeads. While
microdrops of protein delivered onto planar surfaces are the
most familiar format, alternative architectures include CD
centrifugation devices based on developments in microfluidics
[Gyros] and specialised chip designs, such as engineered microchannels
in a plate [The Living Chip™, Biotrove] and tiny 3D posts
on a silicon surface [Zyomyx]. Particles in suspension can
also be used as the basis of arrays, providing they are coded
for identification; systems include colour coding for microbeads
[Luminex, Bio-Rad] and semiconductor nanocrystals [QDots™,
Quantum Dots], and barcoding for beads [UltraPlex™, Smartbeads]
and multimetal microrods [Nanobarcodes™ particles, Nanoplex
Technologies].
Beads can also be assembled into planar arrays on semiconductor
chips [LEAPS technology, BioArray Solutions].
Protein
immobilisation considerations
Variables in immobilisation of proteins include both the coupling
reagent and the nature of the surface being coupled to. The
properties of a good protein array support surface are that
it should be chemically stable before and after the coupling
procedures, allow good spot morphology, display minimal nonspecific
binding, not contribute a background in detection systems,
and be compatible with different detection systems. The immobilisation
method used should be reproducible, applicable to proteins
of different properties (size, hydrophilic, hydrophobic),
amenable to high throughput and automation, and compatible
with retention of fully functional protein activity. Orientation
of the surface-bound protein is recognised as an important
factor in presenting it to ligand or substrate in an active
state; for capture arrays the most efficient binding results
are obtained with orientated capture reagents, which generally
requires site-specific labelling of the protein.
Both
covalent and noncovalent methods of protein immobilisation
are used and have various pros and cons. Passive adsorption
to surfaces is methodologically simple, but allows little
quantitative or orientational control; it may or may not alter
the functional properties of the protein, and reproducibility
and efficiency are variable. Covalent coupling methods provide
a stable linkage, can be applied to a range of proteins and
have good reproducibility; however, orientation may be variable,
chemical derivatisation may alter the function of the protein
and requires a stable interactive surface. Biological capture
methods utilising a tag on the protein provide a stable linkage
and bind the protein specifically and in reproducible orientation,
but the biological reagent must first be immobilised adequately
and the array may require special handling and have variable
stability.
Several
immobilisation chemistries and tags have been described for
fabrication of protein arrays. Substrates for covalent attachment
include glass slides coated with amino- or aldehyde-containing
silane reagents. In the Versalinx™ system [Prolinx],
reversible covalent coupling is achieved by interaction between
the protein derivatised with phenyldiboronic acid, and salicylhydroxamic
acid immobilised on the support surface. This also has low
background binding and low intrinsic fluorescence and allows
the immobilised proteins to retain function. Noncovalent binding
of unmodified protein occurs within porous structures such
as HydroGel™ [PerkinElmer], based on a 3-dimensional
polyacrylamide gel; this substrate is reported to give a particularly
low background on glass microarrays, with a high capacity
and retention of protein function. Widely used biological
coupling methods are through biotin/streptavidin or hexahistidine/Ni
interactions, having modified the protein appropriately. Biotin
may be conjugated to a poly-lysine backbone immobilised on
a surface such as titanium dioxide [Zyomyx] or tantalum pentoxide
[Zeptosens].
Fabrication
Array fabrication methods include robotic contact printing,
ink-jetting, piezoelectric spotting and photolithography.
A number of commercial arrayers are available [e.g. Packard
Biosience] as well as manual equipment [V & P Scientific].
Bacterial colonies can be robotically gridded onto PVDF membranes
for induction of protein expression in situ.
Provided
by Philipp Angenendt and Dolores Cahill, Max-Planck-Institute
of Molecular Genetics, Berlin, Germany
At the limit of spot size and density are nanoarrays, with
spots on the nanometer spatial scale, enabling thousands of
reactions to be performed on a single chip less than 1mm square.
BioForce Laboratories have developed nanoarrays with 1521
protein spots in 85sq microns, equivalent to 25 million spots
per sq cm, at the limit for optical detection; their readout
methods are fluorescence and atomic force microscopy (AFM).
A
microfluidics system for automated sample incubation with
arrays on glass slides and washing has been codeveloped by
NextGen and PerkinElmer Lifesciences.
Detection
Fluorescence labelling and detection methods are widely used.
The same instrumentation as used for reading DNA microarrays
is applicable to protein arrays. For differential display,
capture (e.g. antibody) arrays can be probed with fluorescently
labelled proteins from two different cell states, in which
cell lysates are directly conjugated with different fluorophores
(e.g. Cy-3, Cy-5) and mixed, such that the colour acts as
a readout for changes in target abundance. Fluorescent readout
sensitivity can be amplified 10-100 fold by tyramide signal
amplification (TSA) [PerkinElmer Lifesciences]. Planar
waveguide technology [Zeptosens] enables ultrasensitive fluorescence
detection, with the additional advantage of no intervening
washing procedures. High sensitivity can also be achieved
with suspension beads and particles, using phycoerythrin as
label [Luminex] or the properties of semiconductor nanocrystals
[Quantum Dot]. .
A number of novel alternative readouts have been developed,
especially in the commercial biotech arena. These include
adaptations of surface plasmon resonance [HTS Biosystems,
Intrinsic Bioprobes], rolling circle DNA amplification [Molecular
Staging], mass spectrometry [Ciphergen, Intrinsic Bioprobes],
resonance light scattering [Genicon Sciences] and atomic force
microscopy [BioForce Laboratories].
An
array of 110 different antibodies incubated with various levels
of the fluorescently labelled cognate antigens in a serum
background. (Courtesy Dr Brian Haab, The Van Andel Research
Institute, Grand Rapids, MI, USA)
Capture
arrays
These form the basis of diagnostic chips and arrays for expression
profiling. They employ high affinity capture reagents, such
as conventional antibodies, single domains, engineered scaffolds,
peptides or nucleic acid aptamers, to bind and detect specific
target ligands in high throughput manner.
Ligand
binding molecules
Antibody arrays have the required properties of specificity
and acceptable background, and some are available commercially
[BD Biosciences Clontech, BioRad, Sigma]. Antibodies for capture
arrays are made either by conventional immunisation (polyclonal
sera and hybridomas), or as recombinant fragments, usually
expressed in E. coli, after selection from phage or ribosome
display libraries [Cambridge Antibody Technology, BioInvent,
Affitech, Biosite]. In addition to the conventional antibodies,
Fab and scFv fragments, single V-domains from camelids or
engineered human equivalents [Domantis] may also be useful
in arrays.
The
term 'scaffold' refers to ligand-binding domains of proteins,
which are engineered into multiple variants capable of binding
diverse target molecules with antibody-like properties of
specificity and affinity. The variants can be produced in
a genetic library format and selected against individual targets
by phage, bacterial or ribosome display. Such ligand-binding
scaffolds or frameworks include 'Affibodies' based on Staph.
aureus protein A [Affibody], 'Trinectins' based on fibronectins
[Phylos] and 'Anticalins' based on the lipocalin structure
[Pieris]. These can be used on capture arrays in a similar
fashion to antibodies and may have advantages of robustness
and ease of production (not to mention IP issues).
Nonprotein
capture molecules, notably the single-stranded nucleic acid
aptamers which bind protein ligands with high specificity
and affinity, are also used in arrays [SomaLogic]. Aptamers
are selected from libraries of oligonucleotides by the Selex™
procedure and their interaction with protein can be enhanced
by covalent attachment, through incorporation of brominated
deoxyuridine and UV-activated crosslinking (photoaptamers).
Photocrosslinking to ligand reduces the crossreactivity of
aptamers due to the specific steric requirements. Aptamers
have the advantages of ease of production by automated oligonucleotide
synthesis and the stability and robustness of DNA; on photoaptamer
arrays, universal fluorescent protein stains can be used to
detect binding.
Detection
systems for capture arrays
Figure
prepared by Jonas Jarvius, Uppsala (from Taussig and Landegren,
Targets 2:169-176, 2003)
Protein
analytes binding to antibody arrays may be detected directly
or via a secondary antibody in a sandwich assay. Direct labelling
is used for comparison of different samples with different
colours. Where pairs of antibodies directed at the same protein
ligand are available, sandwich immunoassays provide high specificity
and sensitivity and are therefore the method of choice for
low abundance proteins such as cytokines; they also give the
possibility of detection of protein modifications. Label-
free detection methods, including mass spectrometry, surface
plasmon resonance and atomic force microscopy, avoid alteration
of ligand. What is required from any method is optimal sensitivity
and specificity, with low background to give high signal to
noise. Since analyte concentrations cover a wide range, sensitivity
has to be tailored appropriately; serial dilution of the sample
or use of antibodies of different affinities are solutions
to this problem. Proteins of interest are frequently those
in low concentration in body fluids and extracts, requiring
detection in the pg range or lower, such as cytokines or the
low expression products in cells.
Specificity
and cross-reactivity on capture arrays
The question of cross-reactivity is an important one which
applies to all ligand binders and particularly to antibodies,
being the most popular reagents. While antibodies are thought
of as being highly specific, monoclonals can show unpredictable
cross-reactions which will be revealed by thorough screening.
The ultimate usefulness of individual reagents then depends
on the relative level of cross-reaction and specific reaction.
The use of sandwich assays, in which antibody pairs are used
to bind and detect ligand, may go a long way towards eliminating
the problem, since it is unlikely that both members of the
sandwich will exhibit the same cross-reactivity. Polyclonal
antibodies are emerging as array reagents for protein expression
studies; although they require affinity purification, rabbit
sera are easier to produce than monoclonals, and cross-reactions
may be reduced as a result of heterogeneity. There are ambitious
projects to raise monoclonal antibodies and antisera against
the entire human proteome.
An
important general principle is that, for optimal specificity
where assays are highly multiplexed, it is essential to provide
dual level target recognition, i.e. two levels of specificity
for each locus in the array. Sandwich assays achieve this
with two antibodies, photocrosslinking reduces the cross-reactivity
of aptamers and MS provides definitive label-free protein
identification.
Related
formats
An
alternative to an array of capture molecules is one made through
'molecular imprinting' technology, in which peptides (e.g.
from the C-terminal regions of proteins) are used as templates
to generate structurally complementary, sequence-specific
cavities in a polymerisable matrix; the cavities can then
specifically capture (denatured) proteins which have the appropriate
primary amino acid sequence [ProteinPrint™, Aspira Biosystems].
Another
methodology which can be used diagnostically and in expression
profiling is the ProteinChip® array [Ciphergen], in which
solid phase chromatographic surfaces bind proteins with similar
characteristics of charge or hydrophobicity from mixtures
such as plasma or tumour extracts, and SELDI-TOF mass spectrometry
is used to detection the retained proteins. The ProteinChip®
is credited with the ability to identify novel disease markers.
However, this technology differs from the protein arrays under
discussion here since, in general, it does not involve immobilisation
of individual proteins for detection of specific ligand interactions.
Applications
of capture arrays
(1)
Diagnostics
As diagnostic devices, microarrays exploit the power of multiplexing
simultaneous analyses of different samples and repeated analyses
of the same samples. Diagnostics formats include arrays of
antibodies, as in detection of cytokines [e.g. Zyomyx, Zeptosens,
Molecular Staging, Luminex], and antigens to detect serum
antibodies in screens for infections, autoimmune diseases
and allergies. Highly parallel analysis on arrays will allow
determination of tumour markers in extracts with only a minimum
of biopsy material, creating new possibilities for monitoring
cancer treatment and therapy. Discovery of new autoantibody
specificities is possible by screening patient sera against
arrays of human proteins [Protagen].
While
diagnostic arrays have tended to be of relatively low density
and designed for specific assay purposes, they have high throughput
potential through automated image analysis and microfluidics.
(2)
Proteomics: Protein expression profiling
The quantitative detection of proteins in cells and tissues
and comparison in different conditions (health, disease, differentiation,
drug treatment, etc) is a central aim of proteomics. The array
format is well established for the rapid, global analysis
of nucleic acids, as in the use of oligonucleotide and cDNA
arrays for gene expression (transcription) profiling. However,
mRNA expression data has acknowledged shortcomings as an indicator
of actual protein abundance or dynamics, and moreover reveals
nothing about post-translational modifications or protein-protein
interactions. Two-dimensional gel electrophoresis technology,
on which most proteome profiling is based currently, is also
limited in various ways, particularly in the difficulty of
finding and quantitatively estimating low abundance proteins.
For information about the expression of the proteome, protein
and peptide arrays are becoming major tools and the information
that will be obtained from them in the future will complement
transcriptional data. Capture arrays sensitively and accurately
detect low levels of proteins with minimal technical know-how
on the part of the user and we can expect them to be used
widely to measure differential protein expression. They will
provide a powerful and reliable platform for extending molecular
analysis beyond the limitations of DNA chips. This assumes
that the necessary numbers of antibodies or other capture
reagents of required specificity and affinity can be obtained
against the proteins of interest - at the limit, against the
entire proteome.
A
format for differential protein expression profiling using
antibody arrays is shown in the figure above. A mixture (e.g.
of two tissue extracts) is applied to the array and the analytes
of interest are captured by the specific ligand binders, followed
by detection of binding. Similar to comparison of samples
from normal and diseased tissues on DNA arrays or on 2D gels,
reference and test samples can be labelled with Cy3 and Cy5
fluors, mixed, gel filtered to remove unbound dyes and then
incubated on a chip of arrayed antibodies. Increased or decreased
protein expression is assessed using a scanner and up- or
down-regulated proteins can be identified from the ratios
of the two dyes in the familiar 'traffic light' (red, yellow,
green) system. Here, directly labelled (covalently derivatised)
samples are used, but there are a number of alternative detection
strategies.
Large-scale
protein arrays
Large-scale
functional chips have been constructed by immobilising large
numbers of purified proteins and used to assay a wide range
of biochemical functions, such as protein interactions with
other proteins, drug-target interactions, enzyme-substrates,
etc. Generally they require an expression library, cloned
into E. coli, yeast or similar from which the expressed proteins
are then purified, e.g. via a His tag, and immobilised. Cell
free protein transcription/translation is a viable alternative
for synthesis of proteins which do not express well in bacterial
or other in vivo systems.
Provided
by Claudia Gotthold and Dolores Cahill, Max-Planck-Institute
of Molecular Genetics, Berlin, Germany
Screening
for protein functions and interactions
For detecting protein-protein interactions, protein arrays
can be in vitro alternatives to the cell-based yeast
two-hybrid system and may be useful where the latter is deficient,
such as interactions involving secreted proteins or proteins
with disulphide bridges. High-throughput analysis of biochemical
activities on arrays has been described for yeast protein
kinases and for various functions (protein-protein and protein-lipid
interactions) of the yeast proteome, where a large proportion
of all yeast open-reading frames was expressed and immobilised
on a microarray. Large-scale 'proteome chips' promise to be
very useful in identification of functional interactions,
drug screening, etc. [Proteometrix]. Another possible screen
will be for the effect of polymorphisms arising from disease-related
coding SNPs (SAPs, single amino acid polymorphisms); such
information may be valuable in ascertaining the effects of
SNPs on drug responses and side effects in patients (pharmacogenomics).
One
restriction is that proteins which are only functional as
multicomponent complexes will probably not be analysable on
protein arrays.
Library
screening
As a two-dimensional display of individual elements, a protein
array can be used to screen phage or ribosome display libraries,
in order to select specific binding partners, including antibodies,
synthetic scaffolds, peptides and aptamers. In this way, 'library
against library' screening can be carried out. Screening of
drug candidates in combinatorial chemical libraries against
an array of protein targets identified from genome projects
is another application of the approach.
Challenges
and bottlenecks
There are a number of important technical challenges and bottlenecks
in protein array technologies, some of which are unique to
proteins while others are common to high throughput methods
in general, which will need to be solved in order to achieve
the maximum capability. They include the problems of obtaining
global, functional protein expression for array construction
and selection of ligand binders, aspects of protein coupling
to surfaces, the sensitivity and dynamic range of detection
systems, and standardisation and data storage. At each bottleneck
there are choices of alternative methods. Issues which need
to be addressed include the following.
Protein
production and function
A bottleneck in creating protein arrays, especially those
which aim to be global, is the production (expression and
purification) of the huge diversity of proteins which will
form the array elements, including capture molecules. There
are a number of alternative protein expression systems, including
bacterial, yeast, baculovirus and cell-free. A collection
of high-quality expression clones is required for protein
purification and systems for the production of proteins in
high throughput manner must be developed. The challenge will
be to make expression methods sufficiently comprehensive such
that potentially all proteins become available; different
systems will be employed for different proteins. One aim will
be to express many of the proteins for functional analysis,
and another is to raise antibodies and other capture reagents
against them for array production, immunohistology and chromatography.
Production of nonredundant sets of recombinant full length
proteins from cDNA libraries is greatly improved by Unigene-Uniprotein
sets,
where each gene-protein is uniquely represented.
Provided
by Dolores Cahill, Max-Planck-Institute of Molecular Genetics
A
further consideration for many purposes is that arrayed proteins
should be correctly folded and functional. This will require
extensive and almost individual validation, which with proteins
of unknown function may be hard to achieve! Particular difficulties
relate to production and functional immobilisation of membrane
proteins, which comprise a large proportion of the proteome;
these may be best accessed in the new cell arrays.
Proteome
chemistry is also hugely complicated by the existence of frequent
and varied post-translational modifications (PTMs). The problem
will be how to incorporate PTMs, of which phosphorylation
and glycosylation are just two of many, into protein arrays.
As well as wanting to put PTMs onto the chip surface, we would
also like to know the PTMs of captured proteins, which can
be determined through PTM-specific antibodies or mass spectrometry.
Capture
reagents and cross-reactivity A current limitation on capture arrays is the availability
of the antibodies (or scaffold proteins, etc.), especially
where pairs are required against each target for sandwich
assays. Accessing very large numbers of capture agents is
a major challenge. Automated screening of antibody or scaffold
libraries against arrays of target proteins will be the most
rapid way of developing the thousands of reagents required
for protein expression profiling. There is some discussion
over whether polyclonal antisera, hybridomas or selection
from library systems is the best way forward, but in practice,
providing they are screened thoroughly for cross-reactivity
(below), products of all three are used successfully in the
array format.
The
design of capture arrays, particularly when screening against
heterologous mixtures such as plasma and tissue extracts,
needs to take into consideration the problems of cross-reactivity
which will occur particularly with highly multiplexed assays.
Antibodies can be surprisingly cross-reactive, which in the
high throughput microarray field can render results misleading
or, at worst, useless. Successful multianalyte analysis will
therefore require careful screening of each polyclonal antiserum
or hybridoma clone for cross-reactions against all antigens
on the array. The use of combinations of antibodies against
individual targets in sandwich assays should help to minimise
cross-reactions. The importance of dual level target recognition,
ensuring two levels of specificity for each element in the
array, has been noted. This can be achieved through combinations
of antibodies against individual targets in sandwich assays
or by mass spectrometry to confirm the identity of bound ligands.
Novel detection systems which further increase specificity,
such as proximity ligation, will help to minimise cross-reactions.
Array
technology
What are the best coupling chemistries and supports? There
are several options available in both categories (see Table
above). Comparisons of different systems are gradually becoming
available. The stability and lifetime of protein arrays in
different formats needs to be considered; protein arrays are
likely to be far less robust than DNA arrays.
Detection
methods are another important consideration, with requirements
of sensitivity, accuracy and quantitation over a wide range.
The design of the array will be influenced by the readout
system.
Standardisation
is an issue common to all high throughput technologies: the
existence and development of many alternative formats and
conditions inevitably leads to problems in comparison of results.
Standards for protein arrays and a framework for their implementation
will need to be established at an international level.
Cost
and marketability Currently there is little indication of the relative costs
of the technologies on offer. Although diagnostics would be
a probable driver of the technology, it is not clear whether
companies which currently market immunodiagnostic test kits
will be particularly eager to develop cheaper miniaturised
alternatives.
Comments
on this webpage and suggestions for improvement are welcome.
Please e-mail them to Mike
Taussig.
Page last update 8th December, 2003.
.
A number of novel alternative readouts have been developed,
especially in the commercial biotech arena. These include
adaptations of surface plasmon resonance [HTS Biosystems,
Intrinsic Bioprobes], rolling circle DNA amplification [Molecular
Staging], mass spectrometry [Ciphergen, Intrinsic Bioprobes],
resonance light scattering [Genicon Sciences] and atomic force
microscopy [BioForce Laboratories]. 
