ReviewProgress in protein and antibody microarray technology
Introduction
The basic concept of microarray technology (see Glossary) was initiated by the ambient analyte model of Ekins and colleagues [1, 2, 3], which states that ‘microspot’ assays that rely on the immobilisation of interacting elements on a few square microns should, in principle, be capable of detecting analytes with a higher sensitivity than conventional macroscopic immunoassays. On the basis of such ideas, and boosted by the completion of whole-genome sequencing projects, DNA microarray technology rapidly became the first application of this model [4, 5]. However, the realization that the investigation of genetic information could not provide sufficient insight to understand complex cellular networks, as well as the missing close relationship between mRNA and protein abundance, eventually led to the development of a comparable technology for the analysis of proteins [6, 7, 8]. To achieve this task, antibodies, being natural binders of proteins, were immobilised in an array on a solid support to create antibody microarrays. In parallel, protein microarray technology evolved for the study of protein interactions and modifications. Although such arrays are envisaged to become a valuable tool for tasks such as the characterisation of enzymes [9, 10] or antibody specificity [11, 12], as well as for the elucidation of gene function [13, 14], many limitations of the technology are still unsolved and prevent protein microarray technology from reaching its full potential. These limitations include the generation of content and the conservation of protein functionality during immobilization, as well as the provision of the required absolute and relative sensitivity.
Originally, protein assays were developed in the format of enzyme-linked immunosorbent assays (ELISAs) [15] in microtiter plates with up to 96 wells. Owing to their robustness and sensitivity, ELISAs soon became the gold standard for protein quantification and were adapted to 384-well plates for increased throughput and decreased consumption. One of the first steps to further increase the complexity of ELISA experiments was performed by Mendoza and colleagues [16], who created arrays of 144 elements in each well of a 96-well microtiter plate. This novel approach allowed for multiplex screening of different samples against each array set. One of the next steps in the development of high-content microarrays comprised the production of arrays by high-density spotting of bacteria onto nitrocellulose filters [17]. The bacteria expressed and secreted antibody fragments that were subjected to a filter-based ELISA for the identification of antibody fragments that were specific for the tested antigens. The last step with regard to miniaturisation came along with the spotting of purified proteins and antibodies on coated glass slides. The rigid structure of glass slides allowed an increase in feature density and permitted quantitative assays with diminished amounts of sample solution [18, 19].
As an alternative format to microarrays, the xMAP technology of Luminex Corporation (http://www.luminexcorp.com) is rapidly evolving. In contrast to conventional microarray technology, xMAP does not rely on the spatial separation of capture molecules on glass slides, but instead uses beads for immobilization that are colour-coded by different ratios of two fluorescent dyes. During readout, two different fluorescent signals are recorded, with one signal arising from the fluorescent reporter molecule that monitors the binding event and the other originating from the colour code of the bead for identification of the capture molecule. This combination allows multiplex analysis of up to 100 different species in a liquid environment without any washing steps [20, 21].
Section snippets
Surface coatings
As the objective of protein and antibody microarray technology is the study of interaction partners, the provision of optimal binding conditions is a crucial feature of the microarray support. In previous years PVDF (polyvinylidene fluoride) membranes were the support of choice for high-density protein macroarrays [22, 23] and microarrays [12]. The demand for even higher densities as well as the need for decreased sample consumption and quantification led to the application of glass slides as
Assay conditions and detection
The optimisation of assay conditions is another major challenge for microarray technology. Experience from DNA microarrays has shown that the elucidation of assay conditions that allow optimal binding of all molecules present in the analyte is still quite a challenging task, even for such a uniform molecule as DNA [34]. The transfer of microarray technology to the protein amplifies the problem and becomes crucial with increasing content on the array. Another challenge is the absolute and
Generation of content
One of the key challenges for the success of protein and antibody microarray technology is the provision and generation of content. To date, this step represents a major hurdle, as there is no simple way of generating large and diverse sets of proteins or antibodies.
The complexity of this task is influenced by the application and its requirements with regard to nativity of the immobilised substances. Protein chips are used for a large variety of applications, such as screens of the immune
Recent applications
Although protein and antibody microarray technology are at an early stage of development, several applications in areas such as autoantibody profiling, cancer research or signal pathway characterisation highlight their potential.
In the area of autoimmune profiling, Robinson and colleagues [55] fabricated arrays containing 196 distinct biomolecules, comprising proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. With such arrays, they
Conclusions
The wide variety of different applications in which protein and antibody microarrays are employed reflects the versatility of the technology and underlines the urgent need for technologies that are capable of high-throughput analysis of proteins and antibodies. Although several limitations currently hinder widespread use, it can be expected that advances such as protein production on the chip and the generation of large full-length expression libraries will facilitate the generation of protein
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