Thomas O. Joos

Protein microarray technology.

This review summarizes the major activities in the field of protein microarray technology. A short summary of the theoretical concepts of miniaturized ligand binding assays explains why such microspot assays represent the most sensitive approaches for capture-target assays. The main focus of this review is centered on the applications using miniaturized and parallelized protein binding assays which rely on the product formation between immobilized capture molecules and their corresponding target molecules which are present in the sample. These types of ligand binding assays are useful tools for protein identification, quantification and protein affinity studies. Protein identification and quantification assays have a great potential in the field of diagnostics and proteomics where many different protein markers which are present in complex samples have to be analyzed in parallel. Protein affinity assays can be used to analyze interactions between proteins such as antibodies, receptors or enzymes with other proteins, peptides, low molecular weight compounds, oligosaccharides or DNA. Different applications of protein microarray-based assays and their huge potential for diagnostic and proteomic approaches will be discussed.

A microarray enzyme‐linked immunosorbent assay for autoimmune diagnostics

In order to quantify autoantibodies in the sera of patients with autoimmune disease, we have created a microarray‐based immunoassay that allows the simultaneous analysis of 18 known autoantigens. The microarrays contain serial dilutions of the various antigens, thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. The assay is very sensitive and highly specific: as little as 40 fg of a known protein standard can be detected with little or no cross‐reactivity to nonspecific proteins. The signal intensities observed from serial dilutions of immobilized antigen correlate well with serial dilutions of autoimmune sera. Miniaturized and highly parallelized immunoassays like these will reduce costs by decreasing reagent consumption and improve efficiency by greatly increasing the number of assays that can be performed with a single serum sample. This system will significantly facilitate and accelerate the diagnostics of autoimmune diseases and can be adapted easily to any other kind of immunoassay.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Protein microarrays for diagnostic assays

Protein microarray technology has enormous potential for in vitro diagnostics (IVD). Miniaturized parallelized immunoassays are perfectly suited to generating a maximum of diagnostically relevant information from minute amounts of sample whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first products are already on the market. This article reviews the current state of protein microarrays and discusses developments and future demands relating to protein arrays in their role as multiplexed immunoassays in the field of diagnostics.

Miniaturised multiplexed immunoassays.

Miniaturised immunoassays are of general interest for applications that require the simultaneous determination of different parameters from a minute sample of material. Apart from planar microarray-based systems, bead-based flow cytometric approaches are well suited for the multiplexed detection of target molecules, especially when the number of parameters that have to be determined in parallel is limited.

Pathogen specific cytokine release reveals an effect of TLR2 Arg753Gln during Candida sepsis in humans

Toll-like receptors (TLRs) are crucial pattern-recognition receptors (PRRs) for activation of innate and adapted immunity. TLR2 heterodimerizes with TLR1 or TLR6 to recognize multiple pathogen-associated molecular patterns (PAMPs) of fungi, Gram-positive pathogens, and mycobacteria. Receptor activation culminates in monocyte, T-helper (Th)1, and Th2 cytokine release. Single nucleotide polymorphisms (SNPs) Arg753Gln and Arg677Trp affect TLR2 responsiveness and may contribute to the course of sepsis, which is associated with substantial morbidity and mortality during intensive care treatment. We genotyped 325 critically ill patients with septic shock, and performed a detailed clinical follow-up with 47 of these patients. Here, we investigated whether distinct sepsis episodes result in defined plasma cytokine patterns, and whether cytokine profiles may be linked to the TLR2 polymorphisms. Blood sampling was done daily and microbiological testing was performed on a routine basis. DNA was extracted from whole blood and TLR2 SNPs were typed by pyrosequencing. Cytokines were measured by multiplexed array technologies and the leukocyte phenotype was determined by flow cytometry. Among the 325 ICU patients, 17 individuals (5.2%) were heterozygous for Arg753Gln. The SNP Arg677Trp was not found in any patient. Episodes of Gram-negative, Gram-positive, and Candida sepsis were recorded. During Gram-positive sepsis, the cytokine pattern did not differ between Arg753Gln heterozygous patients and wild type patients. By contrast, during Candida sepsis, the Arg753Gln heterozygous patients showed biomarker patterns that differed from wild type patients with elevated TNF-alpha plasma concentrations, but reduced IFN-gamma and IL-8 levels. In conclusion, TLR2 SNP Arg753Gln results in altered cytokine release in response to Candida but not to Gram-positive sepsis.

Protein microarrays: catching the proteome

After the completion of the human genome sequencing project, DNA microarrays and sophisticated bioinformatics platforms give scientists a global view of biological systems. In todays proteome era, efforts are undertaken to adapt microarray technology in order to analyse the expression of a large number of proteins simultaneously and screen entire genomes for proteins that interact with particular factors, catalyse particular reactions, act as substrates for protein-modifying enzymes and/or as targets of autoimmune responses. In this review, we will summarise the current stage of protein microarray technology. We will focus on the latest fields of application for the simultaneous determination of a variety of parameters from a minute amount of sample. Future challenges of this cutting-edge technology will be discussed.

Protein Microarrays for Personalized Medicine

Abstract Background: Over the last 10 years, DNA microarrays have achieved a robust analytical performance, enabling their use for analyzing the whole transcriptome or for screening thousands of single-nucleotide polymorphisms in a single experiment. DNA microarrays allow scientists to correlate gene expression signatures with disease progression, to screen for disease-specific mutations, and to treat patients according to their individual genetic profiles; however, the real key is proteins and their manifold functions. It is necessary to achieve a greater understanding of not only protein function and abundance but also their role in the development of diseases. Protein concentrations have been shown to reflect the physiological and pathologic state of an organ, tissue, or cells far more directly than DNA, and proteins can be profiled effectively with protein microarrays, which require only a small amount of sample material. Content: Protein microarrays have become well-established tools in basic and applied research, and the first products have already entered the in vitro diagnostics market. This review focuses on protein microarray applications for biomarker discovery and validation, disease diagnosis, and use within the area of personalized medicine. Summary: Protein microarrays have proved to be reliable research tools in screening for a multitude of parameters with only a minimal quantity of sample and have enormous potential in applications for diagnostic and personalized medicine.

New frontiers in microarray technology development

www.sciencedirect.com Within the past decade, microarray-based assays have moved from being technology-driven to application-oriented high-output assay systems. The basic principles of microarray technology were already described in the early 1980s by Ekins’ Ambient Analyte Theory (Ekins RP: Multi-analyte immunoassay. J Pharm Biomed Anal 1989, 7(2):155–168). The driving force behind this theory was the quest for increased sensitivity in the determination of low concentrations of diagnostically important substances, such as hormones. However, developments within the field of microarray technology have been driven by the urgent demand within the field of genomics to provide global analytical tools to process large amounts of information. This could only be accomplished by testing for all possible analytes simultaneously (‘‘Massive parallel testing’’, 2002, ‘‘Chipping Forecast II.’’ Nat Genet, 32(supplement):461–552). The initial format developed by Schena and colleagues was simply spotted sets of cDNAs immobilized in a microarray format that were able to hybridize to fluorescently labeled RNA (Schena et al.: Science 1995, 270(5235):467–470). They could analyze the expression level of approximately a 1000 genes at the RNA level, a significant accomplishment at the time. Fast-forward about a dozen years and the term ‘microarray’ can be found in every area of molecular biology and microarraybased technologies are entering routine applications (see recent review by Hoheisel J: Nat Rev Genet 2006, 7:200–210). Currently, a scientist can take advantage of a wide variety of array platforms containing probes for whole transcriptome analysis, millions of single nucleotide polymorphisms, fragments of genomic DNA, antibodies, cell lysates, purified proteins, tissue sections, and embedded cells. As important as the development of the basic platforms have been, the growth of bioinformatic and statistical approaches that provide the ability to understand and mine the vast amounts of information generated have been equally important and have matured in parallel (for an introduction see review by Quackenbush J: N Engl J Med 2006, 354(23):2463–2472).

Protein microarrays and multiplexed sandwich immunoassays: what beats the beads?

Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently applied for the identification, quantitation and functional analysis of proteins. Protein microarray technology is of major interest for proteomic research in basic and applied biology as well as for diagnostic applications. Miniaturized and parallelized assay systems have reached adequate sensitivity and hence have the potential to replace singleplex analysis systems. However, robustness and automation needs to be demonstrated before this technology will finally prove suitable for high-throughput applications. Miniaturized and parallelized sandwich immunoassays are the most advanced assays formats among the different protein microarray applications. Multiplexed sandwich immunoassays can be used for the identification of biomarkers and the validation of potential target molecules. In this review an overview will be given on the current stage of protein microarray technology with a special focus on miniaturized multiplexed sandwich immunoassays.